Microfluidics for the detection of minimal residual disease in acute myeloid leukemia patients using circulating leukemic cells selected from blood

Microfluidic assay for the selection of circulating leukemic cells from peripheral blood for the early detection of minimal residual disease in acute myeloid leukemia patients.

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

Blood ◽

10.1182/blood.v90.6.2465 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2465-2470 ◽

Cited By ~ 177

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.

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

Blood ◽

10.1182/blood.v90.6.2465.2465_2465_2470 ◽

1997 ◽

Vol 90 (6) ◽

pp. 2465-2470 ◽

Cited By ~ 5

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.

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

Blood ◽

10.1182/blood.v106.11.1461.1461 ◽

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

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Peripheral blood minimal residual disease may replace bone marrow minimal residual disease as an immunophenotypic biomarker for impending relapse in acute myeloid leukemia

Leukemia ◽

10.1038/leu.2015.255 ◽

2015 ◽

Vol 30 (3) ◽

pp. 708-715 ◽

Cited By ~ 41

Author(s):

W Zeijlemaker ◽

A Kelder ◽

Y J M Oussoren-Brockhoff ◽

W J Scholten ◽

A N Snel ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Minimal Residual Disease ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Residual Disease ◽

Minimal Residual ◽

Acute Myeloid

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

Blood ◽

10.1182/blood.v96.12.3948.h8003948_3948_3952 ◽

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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The kinetics of reduction of minimal residual disease impacts on duration of response and survival of patients with acute myeloid leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.6533 ◽

2006 ◽

Vol 24 (18_suppl) ◽

pp. 6533-6533

Author(s):

F. Buccisano ◽

L. Maurillo ◽

G. Del Poeta ◽

M. Del Principe ◽

C. Mazzone ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Minimal Residual Disease ◽

Relapse Rate ◽

Myeloid Leukemia ◽

Residual Disease ◽

Leukemic Cells ◽

Rank Statistic ◽

Multiparametric Flow Cytometry ◽

Minimal Residual ◽

Acute Myeloid

6533 Background: Multiparametric flow-cytometry is frequently used to assess the levels of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients achieving complete remission after intensive chemotherapy. In our previous experience, MRD negativity after consolidation cycle, as defined by a level of bone marrow residual leukemic cells ≤3.5x10−4, was associated with a significantly longer relapse free survival (RFS) and overall survival (OS). The aims of the present study were to confirm, in a larger series of patients, the prognostic relevance of the post consolidation MRD assessment and to validate the threshold of 3.5x10−4. Methods: 100 patients affected with AML were entered into the EORTC/GIMEMA protocols AML10/AML12 (age <61 yrs) or AML13/AML15 (age >61 yrs), consisting in intensive induction and consolidation cycles. Median age was 52 years (range 18–78), all FAB subtypes were represented with the exception of APL cases. A Maximally Selected Rank Statistic analysis was used to select the best threshold. Results: The statistical test confirmed that the level of 3.5×10−4 residual leukemic cells was the most significant in discriminating categories of risk. In fact, levels of MRD measured at the post-consolidation time-point identified 2 distinct subgroups: 36 patients MRD negative and 56 MRD positive. MRD negative patients had a better outcome in terms of relapse rate (P<0.001), OS (P=0.039) and RFS (P=0.008), regardless of MRD status after induction. In fact, patients entering MRD negativity only after consolidation showed the same outcome as those achieving early negativity after induction. The multivariate analysis, including karyotype, age, MDR1 phenotype and post induction and post consolidation MRD levels, indicated that the post-consolidation MRD status was an independent factor affecting relapse rate (P<0.001), OS (P=0.039) and RFS (P=0.008). Conclusions: We conclude that 1) the threshold of 3.5x10−4 is valid in discriminating risk categories in AML; 2) MRD assessment at post-consolidation check-point is critical to predict disease outcome. The incorporation in clinical trials of flow-cytometric MRD determination may allow a more accurate risk stratification of AML patients. No significant financial relationships to disclose.

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Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B–ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease

Blood ◽

10.1182/blood.v99.7.2315 ◽

2002 ◽

Vol 99 (7) ◽

pp. 2315-2323 ◽

Cited By ~ 115

Author(s):

Tomasz Szczepański ◽

Marja J. Willemse ◽

Bas Brinkhof ◽

Elisabeth R. van Wering ◽

Mirjam van der Burg ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Minimal Residual Disease ◽

Myeloid Leukemia ◽

Residual Disease ◽

Patient Specific ◽

Gene Rearrangements ◽

Secondary Acute Myeloid Leukemia ◽

Minimal Residual ◽

Selection Of ◽

Acute Myeloid

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecular studies in 96 childhood precursor-B–ALL at diagnosis and at relapse (n = 91) or at presumably secondary acute myeloid leukemia (n = 5). Clonal Ig and TCR targets for MRD detection were identified in 94 patients, with 71% of these targets being preserved at relapse. The best stability was found for IGK-Kde rearrangements (90%), followed byTCRG (75%), IGH (64%), and incompleteTCRD rearrangements (63%). Combined Southern blot and PCR data for IGH, IGK-Kde, and TCRDgenes showed significant differences in stability at relapse between monoclonal and oligoclonal rearrangements: 89% versus 40%, respectively. In 38% of patients all MRD-PCR targets were preserved at relapse, and in 40% most of the targets (≥ 50%) were preserved. In 22% of patients most targets (10 cases) or all targets (10 cases) were lost at relapse. The latter 10 cases included 4 patients with secondary acute myeloid leukemia with germline Ig/TCR genes. In 5 other patients additional analyses proved the clonal relationship between both disease stages. Finally, in 1 patient all Ig/TCR gene rearrangements were completely different between diagnosis and relapse, which is suggestive of secondary ALL. Based on the presented data, we propose stepwise strategies for selection of stable PCR targets for MRD monitoring, which should enable successful detection of relapse in most (95%) of childhood precursor-B–ALL.

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

Blood ◽

10.1182/blood.v96.12.3948 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3948-3952 ◽

Cited By ~ 172

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

Abstract 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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