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.

scholarly journals Immunophenotyping Investigation of Minimal Residual Disease Is a Useful Approach for Predicting Relapse in Acute Myeloid Leukemia Patients

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2465-2470 ◽  
Author(s):  
J.F. San Miguel ◽  
A. Martı́nez ◽  
A. Macedo ◽  
M.B. Vidriales ◽  
C. López-Berges ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Multidrug Resistance ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Rhodamine 123 ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 × 10−3 residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 × 10−3 residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 × 10−3 leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine-123 assay. Patients with ≥5 × 10−3 residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% ± 24%) than those with less than 5 × 10−3 residual cells (mean, 32% ± 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

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.

scholarly journals Immunophenotyping Investigation of Minimal Residual Disease Is a Useful Approach for Predicting Relapse in Acute Myeloid Leukemia Patients

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2465-2470 ◽  
Author(s):  
J.F. San Miguel ◽  
A. Martı́nez ◽  
A. Macedo ◽  
M.B. Vidriales ◽  
C. López-Berges ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Multidrug Resistance ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Rhodamine 123 ◽  
Minimal Residual ◽  
Acute Myeloid

A high complete remission rate is currently achieved in patients with acute myeloid leukemia (AML). However, many patients eventually relapse due to the persistence of low numbers of residual leukemic cells that are undetectable by conventional cytomorphologic criteria (minimal residual disease [MRD]). Using immunophenotypic multiparametric flow cytometry, we have investigated in sequential studies (diagnosis and follow-up) the impact of MRD detection on the outcome of 53 AML patients that had achieved morphologic remission with standard AML protocols and displayed at diagnosis an aberrant phenotype. Patients were studied at diagnosis with a panel of 35 monoclonal antibodies in triple staining combinations for detection of aberrant or uncommon phenotypic features. According to these features, a patient's probe was custom-built at diagnosis for the identification of possible residual leukemic cells during follow-up. The level of MRD at the end of induction and intensification therapy correlated with the number of relapses and relapse-free survival (RFS). Thus, patients with more than 5 × 10−3 residual cells (5 residual cells among 1,000 normal bone marrow [BM] cells) identified as leukemic by immunophenotyping in the first remission BM showed a significant higher rate of relapse (67% v 20% for patients with less than 5 × 10−3 residual cells; P = .002) and a lower median RFS (17 months v not reached; P = .01). At the end of intensification, with a cut-off value of 2 × 10−3 leukemic cells, AML patients also separated into two distinct groups with relapse rates of 69% versus 32% (P = .02), respectively, and median RFS of 16 months versus not reached (P = .04). In addition, overall survival was also significantly related to the level of residual cells in the marrow obtained at the end of induction and particularly after intensification therapy (P = .008). Furthermore, we have explored whether residual disease was related with the functional expression of multidrug resistance (MDR-1) at diagnosis as assessed by the rhodamine-123 assay. Patients with ≥5 × 10−3 residual leukemic cells at the end of induction therapy had a significantly higher rhodamine-123 efflux (mean, 56% ± 24%) than those with less than 5 × 10−3 residual cells (mean, 32% ± 31%; P = .04). Finally, multivariate analysis showed that the number of residual cells at the end of induction or intensification therapy was the most important prognostic factor for prediction of RFS. Overall, our results show that immunophenotypical investigation of MRD strongly predicts outcome in patients with AML and that the number of residual leukemic cells correlates with multidrug resistance.

Identification of a Set of Seven Genes for the Monitoring of Minimal Residual Disease in Pediatric Acute Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1461-1461
Author(s):  
Daniel Steinbach ◽  
Alexander Schramm ◽  
Angelika Eggert ◽  
Susann Wittig ◽  
Nadine Pfaffendorf ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Minimal Residual Disease ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Wilms Tumor ◽  
Leukemic Cells ◽  
Genome Wide ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract A stepwise approach which combined genome wide expression profiling and a TaqMan realtime PCR based screening was used to identify new markers for the monitoring of minimal residual disease (MRD) in acute myeloid leukemia (AML). Leukemic cells from 52 children with AML were analyzed. Seven genes were identified which are vastly over-expressed in many patients with AML compared to healthy bone marrow: CCL23, GAGED2, MSLN, SPAG6, and ST18 as well as the previously described markers WT1 and PRAME. This set of genes was analyzed in 141 follow-up samples from 25 patients. The expression of all genes decreased to normal levels in patients who achieved a continuous complete remission. Elevated levels of MRD markers were found prior to relapse in 7 out of 10 patients who relapsed. This set of genes should allow a sensitive and specific monitoring of MRD in AML. Notably, some of these markers could also serve as therapeutic targets or might be involved in leukemogenesis. MSLN is already used as a target for immunotherapy in clinical trials in other malignancies. GAGED2 and SPAG6 belong to the family of cancer testis genes which are also studied intensively as targets for immunotherapy. ST18 is a recently discovered tumor suppressor which was not yet described in hematological malignancies. CCL23 is a chemokine that inhibits the proliferation of healthy hematological stem cells. Names, symbols, and geneID of seven MRD markers Gene Symbol Gene Name GeneID CCL23 chemokine (C–C motif) ligand 23 6368 GAGED2 G antigen, family D, 2 9503 MSLN Mesothelin 10232 SPAG6 sperm associated antigen 6 9576 ST18 suppression of tumorigenicity 18 9705 WT1 Wilms tumor 1 7490 PRAME preferentially expressed antigen in melanoma 23532

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.

Identification Of Leukemia-Specific Mutations For Detection Of Minimal Residual Disease In Acute Myeloid Leukemia Using Cell Sorting and Whole Exome Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2575-2575
Author(s):  
Linda Fogelstrand ◽  
Sara Ståhlman ◽  
Tore Samuelsson ◽  
Jonas Abrahamsson ◽  
Lars Palmqvist
Keyword(s):  
Exome Sequencing ◽  
Cell Sorting ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Cells ◽  
Cell Populations ◽  
Whole Exome ◽  
Minimal Residual ◽  
Acute Myeloid

Abstract The introduction of next generation sequencing techniques into the field of leukemia research has revealed that acute myeloid leukemia (AML) is characterized by a limited number of somatic mutations, in most cases single nucleotide variations (SNVs). In addition to providing insight into the pathogenesis of AML, this information can potentially be used for detection of small amounts of leukemic cells in follow-up samples (minimal residual disease, MRD). The aim of this study was to identify leukemia-specific mutations in AML cells that can serve as leukemia-specific MRD-markers. Identification of leukemia-specific mutations was performed using whole exome sequencing of DNA from sorted leukemic cells and comparison with sorted lymphocytes from the same individual. Cells were obtained from 8 cases of AML, age 30-71 years old, from blood samples taken at the time of diagnosis of AML. Cell sorting was carried out by fluorescence activated cell sorter (FACS), where leukemic cells were defined by their FSC and SSC properties and expression of CD45, CD34, CD117, and HLA-DR. Lymphocytes were sorted based on FSC, SSC and CD45 expression. Purity of cell populations were >98% for leukemic cells and >99% for lymphocytes (with undetectable amounts of leukemic cells). Exome sequencing of sorted cell populations was performed on the Illumina platform with HiScanSQ yielding around 4^107reads per sample. Data were quality assessed by FastQC, aligned to the reference human genome, processed for PCR duplicate removal, variant calling with Genome Analysis Toolkit (GATK) package, annotation of variants with ANNOVAR, and verification in Integrative Genomic Viewer. SNVs and short insertions or deletions present in the dbSNP database were excluded and the resulting SNVs and short insertions and deletions with minimum coverage of 10 were compared between leukemic cells and lymphocytes from the same individual. Leukemia-specific heterozygous mutations were defined as present in >40% of the reads in the leukemic cell sample and present in none of the reads from the corresponding lymphocyte sample. By using these rather strict criteria at least three leukemia-specific SNVs were found in each AML case. Leukemia-specific SNVs (with coverage spanning between 10 and 250) were detected in recurrently mutated genes but also in genes not previously reported to be mutated in AML, e.g. CNNM4, GLYAT, NCKAP1L, PPBP, and PRB1. In the case of previously reported recurrently mutated genes in AML, at least one SNV was found in most AML cases. SNVs in recurrently mutated genes were found to be leukemia-specific in most cases, but in some cases, including PRPF40B, ETV6, and EZH2, SNVs were present in a heterozygous pattern in both leukemic cells and in lymphocytes, indicating that they are germ-line mutations. Genes with leukemia-specific insertions or deletions included NPM1, STAG2, RUNX1, and BCOR. The finding of the insertion in NPM1 in two cases was confirmed by detection of the insertion with conventional fragment analysis used in our clinical laboratory. When the same data analysis was used on exome sequencing data of neutrophilic cells and lymphocytes sorted from normal control samples (n=2), no SNVs or short insertions or deletions were found to differ between these two cell populations. Our results show that by using exome sequencing on sorted cell populations with high purity, leukemia-specific mutations can be identified in AML samples already at diagnosis without the need for additional sampling of normal material or access to remission samples. Information on leukemia-specific mutations at diagnosis could provide a basis for detection of MRD in follow-up samples, either by polymerase chain reaction or targeted deep sequencing. Disclosures: No relevant conflicts of interest to declare.

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