Minimal Residual Disease (MRD) Analysis in Acute Myeloid Leukemia (AML) with Favorable Cytogenetics [t(8;21) and inv(16)] by Real Time PCR(RT-PCR) and Flow Cytometry (FC).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2989-2989
Author(s):  
Granada Perea ◽  
Adriana Lasa ◽  
Anna Aventin ◽  
Alicia Domingo ◽  
Neus Villamor ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Good Prognosis ◽  
Rt Pcr ◽  
Free Survival ◽  
End Of Treatment ◽  
The Mean ◽  
Minimal Residual

Abstract Objectives: To analyze MRD in 65 patients (pts) with good prognosis AML: 30 t(8;21) and 35 inv(16), using both FC and RT-PCR, and to investigate the prognostic value of MRD in the pts outcome. Methods: MRD was monitored in CR pts (n=55) by FC in 101 follow-up samples obtained after various cycles of treatment, as follows: 40 post-induction (ind), 30 post-intensification (int) and 31 at the end of treatment (ttm), and by RT-PCR in 76 samples: 31, 23 and 22, respectively. In 35 pts the two techniques were applied at the same time of the ttm. MRD by FC was assessed using fixed combinations of three monoclonal antibodies. AML1/ETO and CBFb/MYH11 were analyzed following the BIOMED protocol. Results: Twenty-seven percent (n=15) of CR pts relapsed: 6 with t(8;21) and 9 with inv(16). The mean MRD by FC was 1.1% after ind, 0.2% after int and 0.1% at the end of ttm. At the end of ttm, the MRD detected by FC in relapsed and not relapsed pts were significativaly different: 0.3% vs 0.08% (p=0.002). By RT-PCR, the mean of fusion transcript copies/ablx104 differed between relapsed and nonrelapsed pts: 2385 vs 122 (p=0.001) after ind, 56 vs 7.6 after int (p=0.0001) and 75 vs 3.3 (p=0.0001) at the end of ttm. Relapses were more commonly observed in those pts with FC MRD level >0.1% at the end of ttm than in pts with ≤0.1%: 50% vs 12% (p=ns); likewise, using RT-PCR, a cutoff level of >10 copies at the end of ttm correlated with high risk of relapse: 80% of pts with RT-PCR >10 relapsed compared to 12% of pts with levels <10 (p=0.009). The overall survival (OS) probability was 86% for pts with CF MRD ≤0.1 at the end of ttm and 0% for pts with MRD >0.1 (p=0.1) and the leukemia free survival (LFS) was 78% and 44%, respectively (p=0.05). For pts with RT-PCR ≤10 at the end of ttm, the OS was 100% and for pts with RT-PCR >10 it was 30% (p=0.007) and the LFS was 87% and 20%, respectively (p=0.001). MRD was identified after ind in 55% of relapsed pts and at the end of ttm in 83% of relapsed pts. Only 1 pt (1/13) with FC MRD <0.1 and RT-PCR <10 at the end of ttm relapsed. For patients in complete remission, the mean copy level of chimeric transcript was higher for pts with t(8;21) than for those with inv(16): 30.2 vs 17.4 (p=0.0001). Comments: In tandem analysis of MRD by FC and RT-PCR could improve MRD detection in AML pts.

Minimal Residual Disease (MRD) Monitoring in CBFB-MYH11 Acute Myeloid Leukemia (AML) Is of Prognostic Relevance for Relapse-Free Survival.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2298-2298
Author(s):  
Andrea Corbacioglu ◽  
Claudia Scholl ◽  
Karina Eiwen ◽  
Lars Bullinger ◽  
Stefan Frohling ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Prognostic Impact ◽  
Consolidation Therapy ◽  
Relapse Free Survival ◽  
Free Survival ◽  
Transcript Levels ◽  
Minimal Residual

Abstract Detection of minimal residual disease (MRD) in acute myeloid leukemia (AML) associated with specific gene fusions is an important tool for the assessment of response to treatment and the individual risk of relapse. The real-time quantitative RT-PCR (RQ-PCR) method allows the quantification of fusion transcript levels at distinct time points during treatment. While in acute promyelocytic leukemia (APL) MRD monitoring has been clearly shown to be predictive for clinical outcome, the prognostic value of MRD in CBFB-MYH11 AML could not consistently been demonstrated yet. Small patient populations and the availability of bone marrow (BM)/peripheral blood (PB) samples at defined time points mainly hamper most studies. We evaluated the prognostic impact of MRD in a large cohort of CBFB-MYH11 AML by RQ-PCR. A total of 44 patients (16–60 years) were treated within one of the AMLSG treatment trials (AMLHD93 n=4, AMLHD98A n=27, AMLSG07-04 n=13). Patient samples (BM and/or PB) were collected at study entry (n=75), during treatment (n=199), and during follow up (n=140). Following high-dose cytarabine (HiDAC) consolidation therapy, patients received a second course of HiDAC (n=25); autologous stem cell transplantation (SCT) (n=13) or allogeneic SCT from a matched related family donor (n=6) depending on the treatment protocol. Median follow up was 22.5 months. Quantitative CBFB-MYH11 fusion transcript expression was measured by RQ-PCR using TaqMan technology. Primers and probes were chosen according to Europe Against Cancer (EAC) standard protocols. Sensitivities ranged from 10−3 to 10−4.Transcript levels at diagnosis ranged from 6208 to 312987 (median 34293.5). There was no prognostic impact of pretreatment transcript levels on relapse free survival (RFS). The ratio of transcript levels after 2 induction cycles and pretreatment levels ranged from 0 to 0.0049; again, this ratio had no impact on RFS. In contrast, during consolidation therapy 63% of the patients became RQ-PCR negative and RFS was significantly superior (RFS after 2 years 75%) compared to RQ-PCR positive patients (RFS after 2 years 32%) (p=0.03). After consolidation, seven of the RQ-PCR negative patients became positive at least in one BM-sample during follow up. Four patients developed transcript levels above 10 and all relapsed, whereas the three patients with transcript levels remaining below 10 are in continuous remission (p=0.0001). In our study, transcript levels during and after consolidation therapy are significantly associated with clinical outcome in CBFB-MYH11 AML. Risk-adapted therapy may be considered for those patients remaining positive during consolidation therapy. The identification of transcript levels above 10 after consolidation therapy might allow early treatment decisions.

Minimal Residual Disease Monitored With Fluorescence In Situ Hybridization (FISH) In CD34+CD38- Population Predicts Clinical Outcome In Core Binding Factor Acute Myeloid Leukemia (CBF-AML)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1356-1356
Author(s):  
Xiaoxia Hu ◽  
Libing Wang ◽  
Lei Gao ◽  
Sheng Xu ◽  
Shenglan Gong ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Residual Disease ◽  
Consolidation Therapy ◽  
Rt Pcr ◽  
Binding Factor ◽  
Minimal Residual

Abstract Acute myeloid leukemia (AML) is generally regarded as a stem cell disease, known as leukemic initiating cells (LIC), which initiate the disease and contribute to relapses. Although the phenotype of these cells remains unclear in most patients, they are enriched within CD34+CD38- compartment. In core binding factor (CBF) AML, the cytogenetic abnormablities are also existed in LIC. The aim of this study was to determine the prognostic power of minimal residual disease measured by fluorescence in situ hybridization (FISH) in flow sorted CD34+CD38- cells (FISH+CD34+CD38- population) at different period during the therapy. Thirty-six patients under 65 years of age with de novo CBF AML and treated with CHAML 2010 protocol were retrospectively included in this study. FISH efficiently identified the LICs (FISH+CD34+CD38-) in the CD34+CD38- population. The last follow-up was March 31, 2013, and the median follow-up was 336 days (range: 74-814 days). 33 patients with complete remission (CR) were eligible for the study, and 23 patients (23/33, 69.7%) with t (8;21) or AML1/ETO, and the remaining (10/33, 30.3%) with inv(16)/t(16;16) or CBFβ/MYH11. Flow-cytometry based FISH (F-FISH) procedure was performed at diagnosis, before every cycle of consolidation therapy, and every 3 months during follow-up. The FISH+ percentage at diagnosis constituting an average of 2.1% (range: 0.01%-27.5%) of the blast cells and 64.6% (range: 14%-87.8%) of the CD34+CD38- cells. Before the consolidation, FISH+CD34+CD38- population was detected in 13/33 (39.4%) patients. At this checkpoint, we have found the existence of FISH+CD34+CD38- population had prognostic value for the end points relapse free survival (RFS, 12% versus 68%, P=.008), and retained prognostic significance for RFS in multivariate analysis. Furthermore, the detection of FISH+CD34+CD38- before consolidation was found to be significantly associated with decreased OS. (11% versus 75%, P=.0005) Minimal residual disease (MRD) detected with F-FISH had a prognostic value at an earlier checkpoint when compared with flow cytometry and RT-PCR. Meanwhile, the concordance of flow cytomety, RT-PCR and F-FISH was investigated in the same patient cohort. 14 (70%) of 20 samples with detectable fusion transcripts by PCR did not have detectable leukemic cells by F-FISH. Therefore, the concordance for PCR and F-FISH was 63.7%. The concordance of FC and F-FISH was 64.3%: in 40 samples MRD was detected by both methods and in 61 samples MRD was ruled out by a negative result with the tests. With further analysis, the discrepancies among MRD detected with different MRD monitoring approaches before consolidation and after the first consolidation therapy contribute to 84% of the disconcordance. In summary, the detection of FISH+CD34+CD38- cells before consolidation therapy was significantly correlated with long-term survival in de novo CBF AML patients. F-FISH might be easily adopted as MRD monitor approach in clinical practice to identify patients at risk of treatment failure from the early stage during therapy. Disclosures: No relevant conflicts of interest to declare.

WT1 Monitoring of Minimal Residual Disease (MRD) in Patients with Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3695-3695 ◽  
Author(s):  
Michele Malagola ◽  
Crisitina Skert ◽  
Enrico Morello ◽  
Francesca Antoniazzi ◽  
Erika Borlenghi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Residual Disease ◽  
Newly Diagnosed ◽  
End Of Treatment ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Background: Although a complete remission (CR) can be achieved in 70-80% of newly diagnosed acute myeloid leukemia (AML) patients, relapses occur in up to the 50% of cases. Thus, minimal residual disease (MRD) monitoring is a major issue for early detection of patients at high-risk of treatment failure and relapse. Aim: to dynamically evaluate WT1 pan-leukemic molecular marker of MRD in patients with AML. Matherial and methods: 107 newly diagnosed AML patients consecutively treated between 2010 and 2013 were monitored with quantitative WT-1 from bone marrow (BM) and peripheral blood (PB) at baseline, after induction, after the first consolidation course, before allogeneic stem cell transplantation (allo-SCT), at the 3rd and the 6th month after transplantation Results: At diagnosis, 104/107 (97%) had increased PB and BM WT1 levels assessed according to the ELN assay. Eighty-eight out of 107 patients (82%) achieved a complete remission (CR) after induction, 30/88 (34%) relapsed during follow up and 24/107 (22%) were addressed to allogeneic stem cell transplantation (allo-SCT). By univariate analysis, PB-WT > 50x10^4/ABL and BM-WT1 > 250x10^4/ABL after induction (PB: p=0.02; BM: p=0.04), after consolidation (PB: p=0.003), at the end of treatment (PB and BM: p=0.001), at 3rd month of follow up (PB and BM: p=0.005) and at 6th month of follow up (PB: p=0.005) were associated with a reduced overall survival (OS). By multivariate analysis, a BM-WT1 > 250 x 10^4/ABL at the end of treatment was significantly associated with a reduced OS. In order to adapt the cut-off of WT1 in our series of patients, we considered WT1 levels as continuous variables and categorized them at approximately the 25th, 50th, and 75th percentile. A cut-off of PB-WT1 > 25x10^4/ABL and BM-WT1 > 125x10^4/ABL at the end of the treatment program was identified as correlated with reduced leukemia-free survival (LFS) and OS (p=0.001). Similarly, and restricting the analysis on the 24 patients allo-transplanted in CR, 8/11 (73%) with pre-transplant PB-WT1 ≥ 5 and 4/13 (31%) with PB-WT1 < 5 relapsed, respectively (p=0.04). The incidence of relapse was higher in AML patients with PB-WT1 ≥ 5 measured at 3rd (56% vs 38%; p=0.43) and 6th month (71% vs 20%; p=0.03) after allo-SCT. Interestingly, 5/5 (100%) patients with pre-transplant PB-WT1 ≥ 5 who never reduced this level at 3rd or 6th month after allo-SCT experienced a disease recurrence. Conclusions: our data, although retrospectively collected, show that WT1 monitoring may be useful to predict the relapse in AML patients. Acknowledgments: This work was supported in part by Banca di Credito Cooperativo di Pompiano e Franciacorta and Lions Club Bassa Bresciana Association. Disclosures Russo: Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy.

Comparison Between RT-PCR and RQ-PCR for Minimal Residual Disease Detection in Acute Promyelocytic Leukemia: The International Consortium on Acute Promyelocytic Leukemia (IC-APL) Experience,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3552-3552
Author(s):  
Ana Paula Alencar de Lima Lange ◽  
Ana Sílvia Gouvea Lima ◽  
Rafael Henriques Jacomo ◽  
Raul AM Melo ◽  
Rosane Bittencourt ◽  
...  
Keyword(s):  
Minimal Residual Disease ◽  
Acute Promyelocytic Leukemia ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Promyelocytic Leukemia ◽  
Rt Pcr ◽  
International Consortium ◽  
The Third ◽  
Minimal Residual

Abstract Abstract 3552 The International Consortium on Acute Promyelocytic Leukemia (IC-APL) is an initiative of the International Members Committee of the ASH that aims to improve the treatment outcome of acute promyelocytic leukemia (APL) patients in developing countries, which was launched in Mexico, Brazil, Chile and Uruguay. The protocol is identical to the PETHEMA-LPA2005, except for the replacement of idarubicin by daunorubicin. In our interim analysis, estimated 2-year overall and disease-free survivals are 80% and 90%, respectively. The 2-year cumulative incidence of relapse was 5.6%. The median follow-up among survivors was 23 months (range: 1 – 56 months). A secondary aim of IC-APL was to establish molecular monitoring for minimal residual disease (MRD) as a standard practice for APL patients in these countries and to use the results obtained to guide therapy. According to the IC-APL protocol, testing for the PML-RARA fusion transcript was to be performed at diagnosis, end of induction (optional), after the third cycle of consolidation, and every 3 months during maintenance. Considering that real-time quantitative polymerase chain reaction (RQ-PCR) provides a number of advantages compared to conventional non-quantitative reverse transcriptase PCR (RT-PCR), we retrospectively compared the results obtained by both techniques. We analyzed 400 bone marrow (BM) samples from 97 patients with de novo APL enrolled in the IC-APL protocol in Brazil. Of the 97 patients, 78 were considered eligible. The mean age was of 35.8 years with 46 males. Among eligible patients, 49 corresponded to bcr1 ; one to bcr2 and 28 to bcr3 subtype of PML breakpoint. To quantify the fusion transcript PML-RARA we used standardized assays developed in the Europe Against Cancer (EAC) program, normalized to the expression of the ABL gene. The results were compared to plasmid standards (Ipsogen, Marseille) and expressed as Normalized Copy Numbers (NCN). Follow-up samples were considered PCR positive when PML-RARA transcripts amplified with Cycle Threshold (Ct) values of ≤40 in at least 2 out of 3 replicates, according to EAC criteria. A total of 71 samples at diagnosis, 50 at the end of induction, 47 after the third consolidation, 202 during maintenance phase and 30 samples after completion of treatment were analyzed. The median NCN of PML-RARA transcripts at diagnosis was 0.5151 and 0.5092 for the bcr1 and bcr3 subtypes, respectively. At the end of induction there was a reduction of about 3 logs (0.0004 for bcr1 and 0.0005 for bcr3). In this phase, six discrepant cases were observed, all presenting positivity by RQ-PCR. None of these cases relapsed and presented consecutive negative results. Considering samples obtained at the end of consolidation, we detected one case of molecular persistence detected by both methods, and two discrepant results, one positive by RT and another by RQ-PCR. Both cases did not relapse. Among samples collected during maintenance and after the end of treatment, two patients (2.5%) relapsed. Both of them were molecular relapses, defined as the detection (in the context of morphological remission) of the PML-RARA transcript by RT-PCR in two consecutive samples collected 15 days apart. RQ-PCR analysis provided much earlier warning of recurring disease, testing positive 5 and 6 months, respectively, before documentation of molecular relapse by conventional RT-PCR assay. Figure 1 show the kinetics of NCN in these two cases. Our results reinforce that the PML-RARA transcript may be detected after induction but this finding was not of prognostic value. However, our study underlines the importance of sequential monitoring to distinguish patients likely to be cured following front-line therapy from those destined to relapse. The RQ-PCR technique was shown to be more sensitive than RT-PCR, providing earlier warning of impending relapse, thereby allowing greater opportunity for successful delivery of pre-emptive therapy. Finally, our results demonstrate that the implementation of the IC-APL allowed the improvement of laboratory standards in parallel to advances in clinical management. Disclosures: Pasquini: Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Pagnano:Novartis: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau.

Droplet Digital PCR Analysis for Diagnosis and Minimal Residual Disease Monitoring in Adult Ph+ Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4989-4989 ◽  
Author(s):  
Nicoletta Coccaro ◽  
Antonella Zagaria ◽  
Luisa Anelli ◽  
Giuseppina Tota ◽  
Paola Orsini ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Fusion Transcript ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Minimal Residual

Abstract Introduction. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are considered an important component of treatment for adult patients affected by Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). In fact, recent studies reported that treating Ph+ ALL with the combination of imatinib and multi-agent chemotherapy improved the overall outcome. Currently, no data are available on the impact of TKIs on minimal residual disease (MRD) in Ph+ ALL. In fact, although the real-time quantitative PCR (RQ-PCR) method, usually employed for monitoring the BCR-ABL1 residual transcript, is sensitive and easy to perform, it lacks a full standardization and international quality validation. Here, we describe a highly sensitive and reproducible droplet digital PCR (ddPCR) test to monitor BCR-ABL1 transcript level in Ph+ ALL. Methods. BCR-ABL1 expression analysis by ddPCR was performed in twenty-two newly diagnosed adult Ph+ ALL patients.The diagnosis was confirmed by qualitative RT-PCR specific for the BCR-ABL1 p190 fusion gene detection. ddPCR experiments were successfully performed in all twenty-two patients at the onset; several follow-up points were evaluated in thirteen patients. ddPCR experiments were performed using primers and probes specific for BCR-ABL1 p190. GUSB was used as control gene. Fifty ng and 750 ng of cDNA templates were used for the onset and for the post-treatment samples, respectively. To increase the limit of detection (LOD), three replicates were run for the post-treatment samples. ddPCR experiments were performed by Bio-Rad's QX200 system and ddPCR data were analyzed with QuantaSoft analysis software (version 1.7.4). Target concentration was expressed as BCR-ABL1 copies/mg. Results. First, we defined the LOD of the BCR-ABL1 p190 ddPCR system, a 10-fold dilution series (100, 10-1, 10-2, 10-3, 10-4, and 10-5) of a pool of p190 positive patients using a diluent-pool of healthy volunteers. This analysis showed remarkable linearity, trueness, and precision down to 10-5. After converting to log-log scale, linear regression showed no concentration-dependent bias, and R2 equaled 0.996. Because the negative samples showed no background, even the detection of a single droplet per well was considered a positive result. The median concentration of the BCR-ABL1 transcript at the onset was 233.8 (min 3.24 - max 1744) x 103BCR-ABL1 copies/mg. Concerning the analysis of follow-up samples, among the thirty-four points that were negative to qualitative nested RT-PCR, twenty-three (68%) resulted to be positive by ddPCR analysis, with a median concentration of 44.95 (min 0.27 - max 573.3) BCR-ABL1 copies/mg. Follow-up points that were negative in ddPCR remained negative even when the experiments were repeated increasing the depth of the analysis, evaluating a total quantity of 4.5 mg of RNA. Conclusions. This study indicates that, as compared to RQ-PCR, ddPCR increases the depth of the quantitative analysis of BCR-ABL1 p190 fusion transcript by allowing the evaluation of larger amounts of RNA. Moreover, our preliminary data revealed that the amount of the BCR-ABL1 fusion transcript at diagnosis is heterogeneous and that the ddPCR is much more sensitive than nested qualitative RT-PCR analysis, as the 68% of samples negative to nested PCR during the follow-up resulted to be positive by ddPCR. Therefore, we suggest that ddPCR represents a precise, sensitive and rapid method for both diagnosis and MRD monitoring of Ph+ ALL patients. Disclosures No relevant conflicts of interest to declare.

Minimal Residual Disease Monitoring In t(8;21) Acute Myeloid Leukemia Based On RUNX1-RUNX1T1 Fusion Quantification On Genomic DNA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1353-1353
Author(s):  
Nicolas Duployez ◽  
Aline Renneville ◽  
Olivier Nibourel ◽  
Alice Marceau-Renaut ◽  
Nathalie Helevaut ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Residual Disease ◽  
Fusion Transcript ◽  
Leukemic Cells ◽  
Sensitivity Threshold ◽  
Pcr Assay ◽  
Dna Amount ◽  
Minimal Residual Disease Monitoring ◽  
Minimal Residual

Abstract Background Acute myeloid leukemia (AML) with t(8;21) chromosomal translocation, leading to the RUNX1-RUNX1T1 fusion, belong to the favorable risk AML subset. However, relapse incidence may reach 30-40% in these patients. Minimal residual disease monitoring (MRD) based on the quantification of RUNX1-RUNX1T1 fusion transcript by real-time quantitative PCR (RQ-PCR) has been reported to be an independent prognostic factor for the risk of relapse. The specificity of the RUNX1-RUNX1T1 fusion and the high sensitivity of RQ-PCR techniques have made RUNX1-RUNX1T1 an ideal marker to assess treatment response in t(8;21) AML. Undetectable MRD could mean either that tumor cells persist in a latent state without RNA expression or that MRD level is below the sensitivity threshold. Studies in chronic myeloid leukemia showed that BCR-ABL DNA was still detectable in patients in long-term complete molecular response with undetectable BCR-ABL fusion transcript. Using a similar approach, we investigated the use of RUNX1-RUNX1T1 DNA as a MRD marker in t(8;21) AML, instead of RUNX1-RUNX1T1 mRNA. This approach allows linking results directly to the amount of leukemic cells, since each leukemic cell contains one copy of the RUNX1-RUNX1T1 sequence, while the level of RUNX1-RUNX1T1 mRNA may vary from a patient to another. Methods This study focuses on 17 patients with t(8;21) AML included in the CBF-2006 trial and for whom frozen material was available for further molecular analysis. Bone marrow and blood samples were collected at AML diagnosis and during follow-up, as defined in the CBF-2006 trial. Eight patients relapsed during follow-up and 9 were still in complete remission at the end of the study. Interestingly, 3 patients relapsed with a previously undetectable MRD (in blood and bone marrow samples). First, we identified the breakpoints in the RUNX1 and RUNX1T1 genes for each patient using long-range PCR approaches, coupled with next-generation sequencing (NGS) on Personal Genome Machine™ (PGM). The stability of the RUNX1-RUNX1T1 rearrangement at relapse was checked by Sanger sequencing. Then, we performed quantification of RUNX1-RUNX1T1 DNA by RQ-PCR using Taqman technology. For each patient, a primer pair and a probe were designed using the patient's unique RUNX1-RUNX1T1 breakpoint sequence. The forward and reverse primers were located in RUNX1 and RUNX1T1 genes, respectively, and the probe was located at the RUNX1-RUNX1T1 junction. Calibration curves were established using 10-fold dilutions of the diagnostic DNA of each patient in normal control DNA. Results were given as a ratio of chimeric DNA amount in the follow-up sample to chimeric DNA amount at diagnosis. Results Chromosomal breakpoints were located in RUNX1 intron 5 for all patients. RUNX1T1 breakpoints were located in intron 1b for 15 patients, and in intron 1a for 2 patients (Fig. 1). Quantification failed for 1 patient which was further leave up. Between 2 and 7 follow-up samples were studied for the other patients (median 4.5). DNA monitoring was strongly correlated with RNA monitoring (Fig. 2). Sensitivity threshold, determined by the lowest diagnostic sample dilution which gives a signal, was 10-5 for 7 patients, 10-4 for 6 patients, and only 5.10-4 for 3 patients. MRD was detectable in 31 samples and undetectable in 30 samples by both methods, whereas MRD was detectable only on RNA in 7 samples, probably because of a lack of sensitivity of the RQ-PCR assay. Interestingly, RUNX1T1-RUNX1 DNA was detected in 3 samples from 2 patients who relapsed and for whom RUNX1T1-RUNX1 transcript was undetectable, despite a good RNA quality. Conclusions Overall, RUNX1-RUNX1T1 MRD levels on DNA and RNA were quite similar. The level of mRNA expression did not seem to change during follow-up when compared with the amount of DNA. MRD monitoring on genomic DNA is a useful method, but with sensitivity variations depending on the patient's breakpoint sequence and the efficiency of the RQ-PCR assay. DNA has potential advantages: it is more stable than RNA and a best substrate for collection, processing, transport and storage. Additionally, interpretation of the results is easier because it is closely related to the number of leukemic cells. However, this method greatly increases complexity, time of implementation, and cost of monitoring MRD, which limits its interest in routine practice. Disclosures: No relevant conflicts of interest to declare.

A Significant Early Detection Of Poor Outcome In Acute Myeloid Leukemia Patients Having a Minimal Residual Disease Using Multiparameter Flow Cytomerty Combined To Mixed Chimerism At Three Months After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4639-4639
Author(s):  
Florence Beckerich ◽  
Mohamad Sobh ◽  
Stephane Morisset ◽  
Adriana Plesa ◽  
Valerie Dubois ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Residual Disease ◽  
Progression Free Survival ◽  
Disease Recurrence ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Free Survival ◽  
Minimal Residual ◽  
Acute Myeloid

Background Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potential curative strategy for acute myeloid leukemia (AML) patients in complete remission (CR) presenting poor prognostic factors or with relapsed/refractory disease. However, the risk for disease recurrence following allo-HSCT remains significant and associated with poor outcomes. After transplantation, the early detection of minimal residual disease (MRD) using immunophenotyping combined to chimerism documentation before morphological relapse may allow for immediate interventions and can lead to better results. Immunophenotyping using Multiparameter Flow Cytometry (MFC) and chimerism documentation by PCR have been widely used to track disease recurrence, although a validated consensus on the use of these techniques in the post-allo-HSCT follow-up has not been established yet. The aim of our study was to evaluate the impact of positive MRD by MFC associated to chimerism documentation at 3 months after allo-HSCT on patients overall and progression-free survival (OS, PFS). Patients and Methods We evaluated 137 AML patients who received allo-HSCT in a single center between January 2005 and October 2012 at our department and for whom a 3 months MRD evaluation and chimerism documentation has been performed using MFC, and PCR analysis. There were 71 (52%) males and 66 females with a median age of 47 years (range: 19-66), 77% had de novo AML, 20% had secondary AML and 3% had biphenotypic AML. According to cytogenetics, 40% were normal, 51% were unfavorable (9% classified as failure). According to molecular markers, 9% were favorable, 31% intermediate, 44% unfavourable and 16% had no molecular markers. At allo-HSCT, 46% of patients were in first complete remission (CR1), 25% were in CR 2 and 29% had active disease; 40% received a full intensity conditioning and 60% got reduced intensity one. As cell source, 35% were bone marrow, 53% peripheral blood and 12% cord blood cells. Donors were related in 53% of the cases (45% were 10/10 HLA matched) and unrelated in 47% of cases (20% were 10/10 HLA matched). MFC was performed using BM samples with a sensitivity of 0.01%. Chimerism analysis was performed on marrow and/or blood samples using polymerase chain reaction (PCR) based on informative polymorphic short tandem repeat with an accuracy of ± 5%, a mixed chimerismwas defined by having 5% or more of recipient cells. Results After transplantation, all patients engrafted, the cumulative incidence of acute GVHD at 3 months was 19.9% (95% CI: 16.2-20.6) while the cumulative incidence of chronic GVHD reached 26.7% (95% CI: 22.9-30.5) at 1 year. After a median follow-up of 16 months (range: 3-77), the median OS was 66 months (65-NR) with a 3 years probability of 64% (95% CI: 56-73), the median PFS was 32 months (13-NR) with a 3 years probability of 50% (95% CI: 37-58) while the transplant related mortality rate reached 13.6% (95% CI: 10-16) at 2 years. The 3 months chimerism evaluation (n=137) showed a mixed chimerism in 12 (9%) patients, while the MFC (n= 62) detected 15 patients with leukemic cells. Sixty eight patients showed morphological relapse after a median time of 4.8 months (1-34.7); the correlation study between MRD positivity, mixed chimerism detection and morphological relapse showed a higher correlation for both chimerism and MFC (correlation=0.69, p<0.001) than if we consider chimerism or MFC alone. Multivariate analysis showed a significant worse OS for patients with 3 months positive MFC [1 year OS of 20% vs. 80%, HR= 4 (95% CI: 1.4-11.7), p=0.01] and patients with mixed chimerism [1 year OS of 21% vs. 70%, HR= 4 (95%CI: 1.3-12.1), p=0.01]; these results were still valid even after stratification on disease status at transplantation. These results applied also in terms of PFS for positive MFC [1 year PFS of 13% vs. 76%, HR= 3.6, p=0.02], and mixed chimerism[1 year PFS of 0% vs. 70%, HR= 7, p=0.001], Figure 1. Conclusion The 3 months MRD evaluation using MFC combined to chimerism documentation seems to be an independent prognostic factor on overall and progression-free survival for AML patients undergoing allo-HSCT. The standardisationof this evaluation may lead to the identification of patients with high relapse risk suggesting the need of early therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Manifold-assisted Reverse Transcription-PCR with Real-Time Detection for Measurement of the BCR-ABL Fusion Transcript in Chronic Myeloid Leukemia Patients

2000 ◽  
Vol 46 (7) ◽  
pp. 913-920 ◽  
Author(s):  
Gisela Barbany ◽  
Anette Hagberg ◽  
Ulla Olsson-Strömberg ◽  
Bengt Simonsson ◽  
Ann-Christine Syvänen ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Real Time ◽  
Minimal Residual Disease ◽  
Reverse Transcription ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Detection System ◽  
Fusion Transcript ◽  
Mrna Quantification ◽  
Minimal Residual

Abstract Background: BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML). Methods: We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system. Results: The detection limit of the method was one positive K562 cell among 105 negative cells. GAPDH was chosen as a reference gene based on the low variation between samples from different stages of the disease and the low signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data. Conclusions: The proposed procedure constitutes a reproducible and sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.

Sign in / Sign up

Export Citation Format

Share Document