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 (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.

Decision-Making At The End Of Consolidation Of Acute Myeloid Leukemia: Negative Effect Of Minimal Residual Disease Detectable With Multiparametric Flowcytometry Combined With Gene Mutations Of FLT3 On Early Relapse

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3921-3921
Author(s):  
Yuichiro Ono ◽  
June Takeda ◽  
Hayato Maruoka ◽  
Yasuhiro Kazuma ◽  
Nobuhiro Hiramoto ◽  
...  
Keyword(s):  
Decision Making ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Gene Mutations ◽  
Decision Making Process ◽  
Consolidation Therapy ◽  
Relapse Free Survival ◽  
Free Survival ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract Background About 70-80% of adult patients (pts) with acute myeloid leukemia (AML) achieve complete remission (CR); however, around a half of them experience a relapse. For the purpose of creating accurate decision-making process of post-consolidation strategy, stratification system using karyotype and recurrent gene mutations have been widely utilized. As is confirmed in childhood acute lymphocytic leukemia, however, the information of minimal residual disease (MRD) status would substantially improve the reliance of decision-making process of adult AML pts. Unfortunately, approximately a half of AML patients lack molecular targets suitable for MRD monitoring. The aims of this study are to evaluate the applicability of MRD detection using multiparametric flow cytometry (MPFC) and to estimate the impact of MRD measured with MPFC at the end of consolidation therapy in improving decision-making process. Patients and Methods We retrospectively studied 81 consecutive pts with newly diagnosed AML who received induction therapies and achieved CR in our institute between January 2007 and March 2013. Pts with acute promyelocytic leukemia were excluded. We routinely analyzed the bone marrow specimens with MPFC for the detection of leukemia-associated immunophenotypes (LAIPs) at diagnosis. Since April 2010, RT-PCR assay examined FLT3-ITD mutation in the same specimens. In pts who had traceable LAIPs, the relationships of the levels of MRD at the end of consolidation therapy with relapse free survival were analyzed. Positive MRD was defined as the detection of 0.2% and more LAIPs-positive cells with MPFC. We compared two patient groups: those with MRD at the end of consolidation (MRDp group) and those without (MRDn group). Relapse-free survival (RFS) was analyzed using the Kaplan-Meier method and the log-rank test was used for comparison between each group. A multivariate Cox regression analysis for RFS was fit to assess the effect of the followings: age at diagnosis (≥ vs. < 65 years old), the number of induction regimens required for achieving CR (≥ vs. < 2 times), cytogenetic risk groups of SWOG (unfavorable vs. favorable/intermediate). Results In 57 / 81 pts, MPFC could detect LAIPs in the bone marrow specimens at diagnosis (70.4% of all subjects; 15-82 years-old; follow-up time [median] 98-2211[517] days). FLT-ITD mutations were found in 13 pts, but not in 39 pts (the remaining 5 pts were not examined). The rate of detection of LAIPs with 6-color MPFC was significantly superior to 3-color MPFC (82.1% vs. 61.0%, p<0.05). Induction chemotherapies the pts received were anthracyclin-containing regimens, such as idarubicin and cytarabin (3+7), in 52 pts (91.2%), low-dose cytarabin-based regimen in 4 pts (7.0%) and azacitidine in 1 pt. (1.8%). The MRDp and the MRDn groups were comprised of 20 and 37 pts (35.1% and 64.9%) , respectively. One-year RFS of the MRDp group was significantly inferior to the MRDn group (28.3% vs. 75.2%; log-rank p<0.0005). In the multivariable analysis using the model above, MRD positivity at the end of consolidation remains a significant predictor (HR, 2.93, 95% CI 1.16-7.45, p<0.05). In addition, the 1-year RFS in the MRDp group with FLT-ITD was significantly shorter than that in the MRDp group without FLT3-ITD (0% vs. 47.6% with positive and negative FLT3-ITD, log-rank p<0.05). In the MRDn group, however, the negative impact of FLT3-ITD was not documented (85.7% vs. 69.3% with positive and negative FLT3-ITD, log-rank p=0.954). Conclusion Our retrospective study confirmed that LAIPs as MRD targets were applicable to the majority of pts with AML; MRD positivity measured with LAIPs was a promising predictor for early relapses at the end of consolidation, as was previously reported. When combined with FLT-ITD status, it might become a more sensitive prognostic factor. Disclosures: Takahashi: celgene: Research Funding.

Prognostic Impact of Minimal Residual Disease inCBFB-MYH11–Positive Acute Myeloid Leukemia

2010 ◽  
Vol 28 (23) ◽  
pp. 3724-3729 ◽  
Author(s):  
Andrea Corbacioglu ◽  
Claudia Scholl ◽  
Richard F. Schlenk ◽  
Karina Eiwen ◽  
Juan Du ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Prognostic Impact ◽  
Consolidation Therapy ◽  
Old Patients ◽  
Minimal Residual ◽  
Acute Myeloid

PurposeTo evaluate the prognostic impact of minimal residual disease (MRD) in patients with acute myeloid leukemia (AML) expressing the CBFB-MYH11 fusion transcript.Patients and MethodsQuantitative reverse transcriptase polymerase chain reaction (PCR) was performed on 684 bone marrow (BM; n = 331) and/or peripheral blood (PB; n = 353) samples (median, 13 samples per patient) from 53 younger adult (16 to 60 years old) patients with AML treated in prospective German-Austrian AML Study Group treatment trials. Samples were obtained at diagnosis (BM, n = 45; PB, n = 48), during treatment course (BM, n = 153; PB, n = 122), and at follow-up (BM, n = 133; PB, n = 183). To evaluate the applicability of PB for MRD detection, 198 paired BM and PB samples obtained at identical time points were analyzed.ResultsThe following three clinically relevant checkpoints were identified during consolidation and early follow-up that predicted relapse: achievement of PCR negativity in at least one BM sample during consolidation therapy (2-year relapse-free survival [RFS], 79% v 54% for PCR positivity; P = .035); achievement of PCR negativity in at least two BM or PB samples during consolidation therapy and early follow-up (≤ 3 months; 2-year RFS, P = .001; overall survival, P = .01); and conversion from PCR negativity to PCR positivity with copy ratios of more than 10 after consolidation therapy. Analysis of paired BM and PB samples revealed BM samples to be more sensitive during the course of therapy, whereas for follow-up, PB samples were equally informative.ConclusionWe defined clinically relevant MRD checkpoints that allow for the identification of patients with CBFB-MYH11–positive AML who are at high risk of relapse. Monitoring of CBFB-MYH11 transcript levels should be incorporated into future clinical trials to guide therapeutic decisions.

Determination of Minimal Residual Disease in Patients with Acute Myeloid Leukemia by Multiparameter Flow Cytometry: Prognostic Impact at Different Checkpoints.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 400-400 ◽  
Author(s):  
Wolfgang Kern ◽  
Daniela Voskova ◽  
Claudia Schoch ◽  
Wolfgang Hiddemann ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Residual Disease ◽  
Multiparameter Flow Cytometry ◽  
Prognostic Impact ◽  
Minimal Residual ◽  
Marrow Cells

Abstract Guiding antileukemic treatment in patients with acute myeloid leukemia (AML) is increasingly based on levels of minimal residual disease (MRD) which can be quantified with high sensitivity by multiparameter flow cytometry (MFC). The optimum checkpoint for determination of MRD during the course of therapy, however, has not yet been determined. We applied MFC using a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIPs) at diagnosis and to quantify MRD by individually selected antibody combinations. The prognostic impact of MRD levels was assessed in comparison to cytogenetics and age. Patients received double induction, consolidation, and maintenance therapies and underwent allogeneic stem cell transplantation if they were younger than 60 years and had a matched related donor. In 286 patients with newly diagnosed and untreated AML MFC-based assessment for the presence of LAIP has been performed. The median percentage of LAIP-positive bone marrow cells at diagnosis was 16.04% (range, 2.54%–76.14%). All individual LAIPs were applied to 26 normal bone marrow samples to estimate sensitivity based on the median percentages of LAIP-positive normal bone marrow cells which ranged from 0.00% to 1.01% (median, 0.02%). A total of 550 follow-up samples has been analyzed in these patients at different checkpoints (CP1, up to day 21 after start of therapy, n=85; CP2, day 22–60, n=122; CP3, day 61–120, n=158; CP4, day 121–365, n=137; CP5, after day 365, n=48). In order to adjust for differences in the percentages of LAIP-positive bone marrow cells at diagnosis the logarithmic difference (LD) between diagnosis and follow-up was calculated for each follow-up sample. The median LDs at the respective checkpoints were: CP1, 2.02; CP2, 2.29; CP3, 2.39; CP4, 2.53; and CP5, 2.81. Separation of patients according to the respective median LDs resulted in differences in event-free survival (EFS; CP1: 21.1 vs. 9.1 months, p=0.0711; CP2: 14.2 vs. 9.3 months, p=0.0095; CP3: 30.9 vs. 13.5 months, p=0.0055; CP4: median not reached vs. 14.1 months, p<0.0001; CP5: median not reached vs. 22.5 months, p=0.0001) and overall survival (OS; CP3: median not reached vs. 21.6 months, p=0.0332; CP4: 90% vs. 53% at 2 years, p=0.0058). Cox analysis using the LDs at the different checkpoints as continuous variables confirmed the prognostic impact on EFS (CP2, p=0.002; CP3, p=0.0003; CP4, p<0.0001; CP5, p<0.0001) and revealed an impact also on OS (CP3, p=0.003; CP4, p=0.001; CP5, p=0.029). Cox regression analysis taking into consideration cytogenetics and age as covariates proved the independent prognostic impact of LD at checkpoints 2 to 5 on both EFS and OS with the exception of LD at checkpoint 2 and OS. In fact, LD at checkpoint 5 was the only parameter independently related to EFS and OS. These data suggest that quantification of MRD by MFC in AML results in powerful and independent prognostic parameters. In particular during the first year of treatment MRD levels provide important prognostic information. Clincal trials should use MRD-based stratification in order to assess the efficacy of early treatment intensification in high-risk AML patients.

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.

High Prognostic Impact of Flowcytometric Minimal Residual Disease Detection In Acute Myeloid Leukemia: Prospective Data From the HOVON/SAKK 42a Study

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 760-760
Author(s):  
Monique Terwijn ◽  
Angèle Kelder ◽  
Wim L.J. van Putten ◽  
Alexander N. Snel ◽  
Vincent H.J. van der elden ◽  
...  
Keyword(s):  
Prognostic Factors ◽  
Risk Stratification ◽  
Myeloid Leukemia ◽  
Poor Prognosis ◽  
Residual Disease ◽  
Risk Groups ◽  
Prognostic Impact ◽  
Induction Cycle ◽  
Minimal Residual

Abstract Abstract 760 Currently, the most important prognostic factors for acute myeloid leukemia (AML) include molecular aberrancies and karyotype of the leukemic blasts. Although these factors have showed to be of utmost importance in upfront risk stratification in current treatment schedules, the treatment outcome of patients within as such defined risk groups is still quite heterogeneous. Therefore, there is an unmet need for therapy-dependent prognostic factors which can be implemented into risk-adapted treatment strategies. Minimal residual disease (MRD) frequency is such a parameter. MRD cells are considered responsible for the outgrowth of AML after treatment, leading to a relapse in 30–40% of the patients in complete remission (CR). In this study, we are the first to report prospective multicenter data on the prognostic impact of MRD frequency in adult AML. In our retrospective study (N.Feller et al. Leukemia 2004), we explored which cut-off points for percentage of MRD cells would define MRD positive (levels above cut-off, MRD+) patients with a relatively poor prognosis, from MRD negative (levels below cut-off, MRD-) patients who showed a longer overall and relapse-free survival (OS and RFS). In search for the most optimal cut-off level which can be used for clinical purpose in risk stratification-directed therapy, we used these cut-offs to evaluate the prognostic value in the current prospective setting. Diagnosis and follow-up samples were collected of 462 patients treated uniformly according to the HOVON/SAKK42a protocol (www.hovon.nl) and MRD frequency was assessed blindly without knowledge of clinical course. MRD detection was accomplished by immunophenotyping by flow cytometry (FCM) through aberrant expression of markers on AML blasts. Together with the expression of normal immature cell markers and/or myeloid lineage markers, this offers a leukemia associated phenotype (LAP). Each LAP was individually designed for each patient in diagnosis bone marrow (BM) or peripheral blood. Subsequently, BM samples obtained during follow-up were analysed for the presence of LAP-positive cells. MRD frequency was expressed as a percentage of leukocytes. The median MRD frequencies of patients in clinical CR after first induction cycle (n=164), second induction cycle (n=182) and consolidation (n=121) were 0.040%, 0.022% and 0.020%, respectively. The cut-off levels for MRD frequency as defined retrospectively were all significant in the identification of patients with adverse (MRD+) and favourable (MRD-) OS and RFS, respectively. After the first cycle, the most significant cut-off was 0.8%, leading to 17 MRD+ patients who showed a median RFS of only 8.6 months, while 147 MRD- patients had a median RFS of >47 months (p=0.003,A). The relative risk of relapse (RR) was 2.9 (95% c.i. 1.4–6.0, p=0.004). After the second induction cycle, a cut-off level of 0.06% was most significant. Above this cut-off, 49 patients showed a median RFS of 7 months, while 133 MRD- patients showed a RFS of more than 47 months (p<0.00001, fig B). The RR was 3.2 (95% c.i. 2.0–5.0, p<0.00001). After consolidation therapy, 11 MRD+ patients with extremely poor prognosis were identified (median RFS 7.3 months vs. >47 months for 110 MRD- patients, p<0.00001, fig C), with a RR of 10.6 (95% c.i. 4.9–22.8, p<0.00001). Multivariate analysis was performed with conventional prognostic factors for AML: cytogenetic risk groups and time to achieve CR. After every cycle of therapy, MRD frequency was an independent prognostic factor for RFS after all cycles (1st cycle: p=0.010, 2nd cycle and consolidation p<0.00001) and for OS after 1st (p=0.023) and 2nd induction cycle (p=0.010). In this prospective multicenter study, already after first induction cycle, MRD detection by FCM was an independent significant factor in the identification of poor prognostic patients. In future treatment studies, risk stratification, e.g. for allogeneic stem cell transplantation, should not only be based on risk estimation determined at diagnosis, but also on MRD frequency as a therapy-dependent prognostic factor. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

Minimal Residual Disease in Patients with Acute Myeloid Leukemia Quantified by Multiparameter Flow Cytomety and Quantitative RT-PCR Is an Independent Prognostic Parameter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 319-319 ◽  
Author(s):  
Wolfgang Kern ◽  
Claudia Schoch ◽  
Torsten Haferlach ◽  
Daniela Voskova ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Prognostic Impact ◽  
Cox Regression Analysis ◽  
Minimal Residual ◽  
Acute Myeloid

Quantification of minimal residual disease (MRD) is becoming increasingly important to guide therapy in patients with acute myeloid leukemia (AML). While MFC can be applied to more patients with AML than QPCR, the latter has the advantage of a higher sensitivity in many cases. We compared data obtained by both methods in parallel in bone marrow samples in 160 patients at diagnosis and at 469 follow-up checkpoints. MFC was applied at diagnosis with a comprehensive panel of antibodies to identify leukemia-associated aberrant immunophenotypes (LAIP) useful for MRD monitoring. QPCR targeted on the leukemia-specific fusion transcripts AML1-ETO, AML1-EVI1, CBFB-MYH11, MLL-AF10, MLL-AF6, MLL-AF9, MLL-ELL, MLL-ENL, and PML-RARA as well as overexpression of EVI1, length mutations of FLT3, and partial tandem duplications of MLL. In order to adjust for differences in the percentages of bone marrow cells covered by the respective LAIP by MFC at diagnosis and for the heterogeneity of transcript levels detected by QPCR at diagnosis, the logarithmic difference (LD) was calculated for each follow-up sample in comparison to the diagnostic sample. There was a significant correlation between MFC and QPCR with regard to the LD from diagnosis to follow-up checkpoint (r=0.645, p=0.000001). Concordant results with regard to negativity between QPCR (no signal) and MFC (<0.01% positive cells) was found in 301/469 (64.2%) samples (both methods positive, 270 (57.6%); both methods negative, 31 (6.6%)). In 44 samples (9.4%) QPCR detected positivity and MFC negativity while in 124 samples (26.4%) MFC detected positivity and QPCR negativity (sensitivity of QPCR was lower than 1:100,000 in some cases). In 133 patients clinical follow-up data was available allowing the analysis of the prognostic impact of MRD levels. Cytogenetics were favorable, intermediate, and unfavorable in 86, 30, and 17 cases, respectively. Median age was 46 years (range, 17–83). Median event-free survival (EFS) was 22.1 months, overall survival (OS) at three years was 77%. The median LDs for MFC and QPCR at the checkpoint 1 (up to day 21), 2 (day 22–60), 3 (day 61–120), 4 (day 121–365), and 5 (after day 365) were 2.40 and 0.62, 2.05 and 1.55, 2.51 and 3.34, 2.71 and 3.70, and 2.60 and 3.45, respectively. Separating patients according to these median LDs resulted in a better EFS and OS for cases with higher LDs at all five checkpoints for each method. Significant differences in EFS were observed at checkpoints 2 (MFC, 22.1 vs. 12.6 months, p=0.0379; QPCR, median not reached vs. 9.9, p=0.0081), 3 (QPCR, 30.9 vs. 14.1 months, p=0.0011), 4 (MFC, median not reached vs. 16.9 months, p=0.0007; QPCR, median not reached vs. 15.1 months, p=0.0102), and 5 (QPCR, median not reached vs. 17.2, p=0.0008). Cox regression analysis taking into consideration cytogenetics, age, WBC count, and bone marrow blast count at diagnosis identified the LD at checkpoint 4 determined by MFC and the LD at checkpoints 2 and 5 determined by QPCR as independent prognostic factors. The results of our analyses confirm that both MFC and QPCR are highly sensitive methods capable of quantifying MRD in AML. While data are concordant for both methods in many cases, either of the two has advantages in distinct cases depending on the individual MRD marker. Clinical trials should consider MRD monitoring by both methods in order to prove their respective roles in risk prediction and treatment stratification.

Minimal Residual Disease After Allogeneic Stem Cell Transplantation: A Comparison Among Multiparameter Flow Cytometry, Wilms Tumor 1 Expression and Chimerism Status (Complete vs Low-Level Mixed Chimerism) in Patients with Acute Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1971-1971
Author(s):  
Giovanni Rossi ◽  
Angelo Michele Carella ◽  
Maria Marta Minervini ◽  
Lucia Savino ◽  
Andrea Fontana ◽  
...  
Keyword(s):  
Residual Disease ◽  
Leukemic Cells ◽  
Mixed Chimerism ◽  
Multiparameter Flow Cytometry ◽  
Relapse Free Survival ◽  
Free Survival ◽  
Low Level ◽  
Chimerism Analysis ◽  
Minimal Residual

Abstract Abstract 1971 Introduction. Relapse represents the main cause of treatment failure after allogeneic stem cell transplantation (allo-SCT). Thus, monitoring of minimal residual disease (MRD) in allografted patients allows an early detection of recurrence and a subsequent intervention prior to florid relapse. Multiparameter Flow Cytometry (MFC), chimerism, cytogenetics and molecular analysis have been widely used for this purpose, although the gold standard needs to be established yet. The evaluation of fusion transcripts represents the most sensitive method but more than 65% of patients do not demonstrate a molecular target. WT1 mRNA is over expressed in > 90% of patients with acute myeloid leukemia (AML) but it is not so expressed in acute lymphoblastic leukemia (ALL). On the MFC analysis instead, the complexity of maturational patterns and phenotypic shifts after therapy may underestimate residual leukemic cells. Finally, previous papers reported that a state of mixed chimerism (MC) in RT-PCR may identify higher risk of relapse in patients with AML. However, the test has a low sensitivity (from 0,1% to 1%) and recipient cells are not necessarily linked to disease recurrence. Hypothesizing the presence of blast cells in the low-level mixed chimerism (MC, 1%<MC<5% of autologous cells) but not in the complete chimerism (CC, no evidence of autologous cells) status, we used these two ranges as markers of positive and negative MRD, respectively. The aim of our study was twofold. Firstly, to assess the overall agreement among chimerism, MFC and WT1 mRNA methods in monitoring MRD. Secondly, to investigate whether such methods were associated to patient' s relapse-free survival. Methods. Fresh BM samples from 24 patients (17 AML and 7 ALL) in both morphological CR and CC or low-level MC status after allo-SCT were investigated. MRD with MFC, WT1 mRNA expression and chimerism analysis was evaluated at different time points: +1, +3, +6,+12,+18,+24 months after allo-SCT. The immunophenotypic analysis was performed using a six-color combinations and acquiring 250.000 events. RQ- PCR to test WT1 mRNA expression was made according to the standardized and quality-controlled method. Chimerism studies were performed with a multiplex amplification of 16 (STR). The agreement between two methods in monitoring MRD after allo-SCT was assessed by Kappa statistic. Moreover, time-to-event analyses were performed using Cox proportional-hazard models with time-dependent covariates. Risks were reported as hazards ratios (HR) along with their 95% confidence interval (95% CI). Results. Comparisons among results of MFC, RQ-PCR for WT1 mRNA and Chimerism were performed in all 67 serial samples obtained from 24 patients. A significant moderate agreement between MFC and WT1 mRNA evaluations was found (k= 0.463, p<0.001) as well as fair agreement between chimerism and MFC (k= 0.284, p=0.009) and chimerism and WT1 mRNA (k= 0.197, p=0.073). These results suggested a concordance among the three investigated techniques. In particular, the low-level MC would well detect the presence of leukemic cells, since the proportion of positive samples for MFC was not statistically different to the proportion of positive samples for WT1 mRNA within samples with low-level MC. Indeed, among 12 samples with low-level MC, 9 samples (75.0%) were also positive for MFC and 7(58.3%) were also positive for WT1 mRNA. Cut-off of 0.1% and 0.01% for MFC, 83.5 × 104 ABL for WT1 mRNA and 96%-99% of donor cells for chimerism were selected. The median follow-up times for relapse-free was 12.8 mths and the overall estimated relapse-free survival after 36 mths was 66.5%. At the end of follow- up, 5 patients relapsed and 4 patients died. Although not significant, the detection of a positive MRD for all methods were associated to a higher incidence of relapse than the negative MRD. Similar HRs were observed in the analysis of MFC(HR = 6.55, 95%CI= 0.71–60.17, p=0.096) and WT1 mRNA (HR= 7.17, 95% CI=0.77–66.42, p=0.083) whilst a slighter HR was found for the chimerism analysis (HR= 0.40, 95%CI= 0.04–4.44, p=0.456). Conclusions. According to our study MFC, WT1 mRNA and CC or low-level MC displayed an overall agreement in monitoring MRD. In particular, the agreement on the low-level MC may suggest the presence of leukemic cells. The detection of a positive MRD by MFC and WT1 mRNA similarly identified higher risk of relapse in patients with acute leukemia (AL) undergone to allo-SCT. 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.

Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3948-3952
Author(s):  
Adriano Venditti ◽  
Francesco Buccisano ◽  
Giovanni Del Poeta ◽  
Luca Maurillo ◽  
Anna Tamburini ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Short Duration ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Relapse Free Survival ◽  
Free Survival ◽  
Minimal Residual ◽  
Acute Myeloid

We used flow cytometry to quantify minimal residual disease (MRD) in 56 patients with acute myeloid leukemia (AML) expressing a leukemia-associated phenotype. Thirty-four patients aged 18 to 60 years were entered into the AML-10 protocol (induction, consolidation, and autologous stem-cell transplantation [ASCT]), whereas 22 patients older than 60 years received the AML-13 protocol (induction, consolidation, and consolidation II). After induction, the level of MRD that was best associated with treatment outcome was 4.5 × 10−4 residual leukemic cells. However, the outcome in patients with at least 4.5 × 10−4 cells (n = 26) was not significantly different from that in patients with fewer leukemic cells (n = 30); there were 15 (58%) relapses in the first group and 12 (40%) relapses in the second. After consolidation, the most predictive MRD cutoff value was 3.5 × 10−4cells: 22 patients had an MRD level of 3.5 × 10−4 cells or higher and 17 (77%) of these patients had relapse, compared with 5 of 29 patients (17%) with lower MRD levels (P < .001). An MRD level of 3.5 × 10−4 cells or higher after consolidation was significantly correlated with poor or intermediate-risk cytogenetic findings, a multidrug resistance 1 (MDR1) phenotype, short duration of overall survival, and short duration of relapse-free survival (P = .014, .031, .00022, and .00014, respectively). In multivariate analysis, this MRD status was significantly associated with a high frequency of relapse (P < .001) and a short duration of overall (P = .025) and relapse-free survival (P = .007). ASCT did not alter the prognostic effect of high MRD levels after consolidation: the relapse rate after transplantation was 70%. Thus, we found that an MRD level of 3.5 × 10−4 cells or higher at the end of consolidation strongly predicts relapse and is significantly associated with an MDR1 phenotype and intermediate or unfavorable cytogenetic findings.

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