scholarly journals Next Generation Flow for Multiple Myeloma Minimal Residual Disease: Igh Rearrangement NGS Is Complement to the NGF

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5609-5609
Author(s):  
Sung-Min Kim ◽  
Jung-Ah Kim ◽  
Dajeong Jeong ◽  
Jiwon Yun ◽  
Kyu Min Lim ◽  
...  
Keyword(s):  
Treatment Response ◽  
Minimal Residual Disease ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Initial Diagnosis ◽  
The Other ◽  
Detection Sensitivity ◽  
Igh Rearrangement ◽  
Minimal Residual

Abstract Background: Detection of leukemia-associated aberrant immuno-phenotype is used to assess minimal residual disease (MRD) by multi-parameter flow cytometry (MFC). However, detection of MRD by MFC remains to be a challenging due to the possible change in aberrant immunophenotype during disease progress. In our present study, we compared International Myeloma Working group (IMW) treatment response and NGF MRD, including BM PC% and cytogenetics. Thereon, we conducted IgH rearrangement study by NGS in cases showing discrepant results. Methods: A total of 35 BM (35 myeloma patients at follow-up) was enrolled. We performed NGF using 8-color antibody panel using Navios flow cytometer and Infinicyt. Linearity of NGF was validated with myeloma cell line (U266) and BM specimen at initial diagnosis in myeloma patient. IgH rearrangement NGS was performed using Immunoseq assay (Adaptive Biotechnologies, Seattle, WA, USA). Paired specimen at initial diagnosis BM and follow-up BM were subjected to NGS study. Results: Detection sensitivity of NGF was <0.001%. Patients who achieved CR or sCR showed MRD negativity in 63.6% (7/11). Twenty-three patients showed neoplastic PCs above LLOQ and their response criteria were 1 sCR, 3 CR, 2 VGPR, 3 PR, 1 MR, 5 SD, 3 progressive disease, 3 relapse, and 2 with unavailable response. Four patients who did not achieve CR (1 VGPR, 1 PR, 1 MR, and 1 SD) showed MRD negativity by NGF. In 4 patients with discrepancy between IMW treatment response and NGF, we compared the results of IgH NGS at initial BM with those after treatment. NGS revealed a persistence of residual clone in 1 patient and an acquisition of new clone after treatment. One patient had same dominant clone both initial diagnosis BM (95.2%; proportion of clone) and follow-up BM (45.8%). The other patient had newly appeared clones in follow-up BM (6.12%, 5.63%, 3.42%, 3.11%, 3.09%) which clones were absent in initial diagnosis BM. The other 2 patients showed heterogeneous clones without dominant clone at follow-up BM by NGS. Results of FISH and immunofixation are summarized in Table 1. This results show IgH rearrangement NGS can detect malignant clone that could not be identified by using NGF. Conclusions: Thirty-six percent of patients who did not achieve CR showed NGF MRD negativity and NGS revealed residual clones in half of them. Switching of immunophenotypes of neoplastic PC can escape monitoring of NGF, and complementary NGS test is needed to catch such drifting clones for monitoring of MRD in MM. Disclosures No relevant conflicts of interest to declare.

Monitoring of Minimal Residual Disease in NPM1 Mutated Acute Myeloid Leukemia (AML) – a Single Center Experience in 27 Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4672-4672
Author(s):  
Dana Dvorakova ◽  
Zdenek Racil ◽  
Ivo Palasek ◽  
Marketa Protivankova ◽  
Ivana Jeziskova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Npm1 Mutation ◽  
Mgb Probe ◽  
Minimal Residual

Abstract Abstract 4672 Background Mutations within NPM1 gene occurs in about 60% of adult cytogenetic normal AML (CN-AML) and represent the single most frequent molecular aberration in this subgroups of patients. These mutations usually occur at exon 12 and induce most frequently a net insertion of four base pairs. Aims To examine the applicability and sensitivity of DNA-based real-time quantitative polymerase chain reaction (RQ-PCR) with mutation-specific reverse primers and common minor groove binding (MGB) probe and to evaluate whether minimal residual disease levels are of prognostic relevance in CN-AML patients with NPM1 mutations. Methods Patients were treated within different AML trials and follow-up samples of peripheral blood or bone marrow were referred to perform an RQ-PCR. Samples were analysed at diagnosis, during, and after therapy. The NPM1 mutations were A (17 pts), B (1 pt), D (2 pts) and 7 patients with individual rare types. For all cases, levels of minimal residual disease were determined by DNA-based RQ-PCR with mutation-specific reverse primer, one common forward primer and one common MGB probe. The NPM1 mutation value was normalized on the number of albumin gene copies and expressed as the number of NPM1 mutations every 106 genomic equivalents. This assay is highly specific as no wildtype NPM1 could be detected. Maximal reproducible sensitivity was 10 plasmide molecules per reaction. Results A total of 950 samples of bone marrow and/or peripheral blood from 27 patients have been analyzed. Twenty of 27 patients (74%) achieved molecular remission (MR), twenty-six of 27 patients (96%) achieved hematological remission (HR). 6 of 27 (22%) patients achieved HR without MR and one patient failed therapy. 8 of 20 patients (40%) with MR after treatment relapsed at molecular level and except one in all these patients hematological relaps occured (one patient is still in HR with bone marrow blast present, but < 5%). Considering relapsed patients, time from molecular to hematological relapse was 1 to 5 months (median: 3 months). Considering all 14 patients with HR without MR (6 pts) or with molecular relapse (8 pts), in 11 of them hematological relaps occured (79%) and molecular positivity anticipating hematological relaps with median of 3,5 month (1-7 months). 3 of these 14 patients are still in HR. Conclusions Mutations within NPM1 gene are a sensitive marker for monitoring minimal residual disease in CN-AML patients. RQ-PCR using a MGB probe is an efficient approach to long-term follow-up of residual leukemia cells and frequent quantitative monitoring is useful for reliably predicting hematological relapse. Achievement of negativity appears to predict favorable clinical outcome. This work was partially supported by research grant No. MSM0021622430 Disclosures: No relevant conflicts of interest to declare.

Minimal Residual Disease In Adult AML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4969-4969
Author(s):  
Ihab A. Eldessouki ◽  
Ola Khorshid ◽  
Eman Kandeel ◽  
Nasr Lahloubi
Keyword(s):  
Treatment Response ◽  
Minimal Residual Disease ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Tumor Burden ◽  
Multiparametric Flow Cytometry ◽  
Post Induction ◽  
Minimal Residual

Abstract Background The achievement of complete hematologic remission (CR) is used as predictor for treatment response in patients with myeloid leukemia (AML).However <5% blasts in the bone marrow does not reflect the presence of tumor burden precisely. Minimal residual disease (MRD) in the first complete remission (CR1) may play a critical rule in assessment of treatment response and prediction of subsequent relapse. Patients and Methods Leukemia associated immunophenotyping (LAIP) for 73 patients with denovo AML monitored at diagnosis , day 14 and day28 post-induction by multiparametric flow cytometry (MFC). Results CR achieved in 60(82%) patients and 13(18%) patients did not. Among the 60(80%) patients who achieved CR 9 (15%) were MRD negative and 51(85%) were MRD positive at day14. Significant association between MRD detection and disease free survival (DFS) using 0.01% cut off value (P=.015). Day 28 post induction show highly significant association between MRD and DFS using 0.01% cut off value (P=0.001) as 38(63%) patients were MRD negative and (27%) were positive. Significant association between MRD detection and overall survival (50 month) at day 14 and day 28 (P=0.02, P=0.001) respectively using cut off value 0.01%. MRD was positive in 63(86%) at day 14 and (37%) at day 28. Conclusion MRD detection at day28 and d14 at the end of induction in patients in CR may have a prognostic significance on clinical outcome and may thus be a useful marker for risk stratification. Disclosures: No relevant conflicts of interest to declare.

Minimal Residual Disease Stratification in Fludarabine Ccyclophosphamide Rituximab (FCR) Therapy in Chronic Lymphocytic Leukaemia (CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1791-1791
Author(s):  
Amjad Hayat ◽  
David O'Brien ◽  
Fiona Quinn ◽  
Fionnuala Keane ◽  
Imelda Parker ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukaemia ◽  
Minimal Residual Disease ◽  
Lymphocytic Leukaemia ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Somatic Hypermutation ◽  
Disease Stratification ◽  
Grade 3 ◽  
Minimal Residual

Abstract Abstract 1791 Six courses of FCR is considered standard therapy for fit, non-(17p) deleted patients with Chronic Lymphocytic Leukaemia (CLL), however the optimal dose of Rituximab in combined chemotherapy for CLL or the number of FCR courses required has never been formally assessed. Minimal residual disease (MRD) eradication (assessed by 6 colour flow-cytometry(FC) is associated with an increased disease free interval and is a logical endpoint in determining length/efficacy of treatment. Serial MRD testing following therapy provides an objective test of disease re-emergence. A multi-centre prospective phase II study in treatment-naïve CLL patients with modified FCR (Rituximab 375mg/m2) using MRD to determine treatment length/efficacy and identify disease emergence recruited from 2009–2012. Standard pre-treatment assessment plus C-T scan, FISH and Ig somatic hypermutation (SHM) analyses were performed. Patient in radiological and MRD-ve remission after 4 courses of FCR stopped therapy and the remainder received 6 courses. All were followed by 6 monthly MRD assessment. Fifty-two patients were included (35M/17F), mean age 52 years (range 37–72), mean WCC 51 × 109/L (range 8–386), elevated LDH 23 of 45 (51%), lymphadenopathy 43 (83%) and hepatosplenomegaly in 37 (71%). Abnormal FISH results were, del(11q) in 15, +12 in 5, del(13q) in 18; SHM 15(29%) mutated and 37(71%) unmutated or with V3–21 gene usage. Post-treatment MRD is available in 43 patients; 36 (70%) were MRD –ve including 18 (42%) after 4 courses. Nine patients did not complete treatment (toxicity −7, progression-1, non-compliance-1). With a mean follow-up of 20.7 months (range 4.5–38), 6 patients have reverted to MRD +ve at a mean of 13 months (range 10–19) from therapy; only the patient with primary treatment failure has required further chemotherapy. Myelotoxoicty resulted in 23 NCI grade 3 episodes and 7 treatment delays of a mean duration of 25 days (range15–32). One further grade 3 toxicity of pneumonia was identified. Modified FCR was effective in this patient cohort with high risk features (38% unfavourable FISH, 71% unfavourable SHM), with 70% patients achieving MRD-ve status on completion of therapy. 42% of patients became MRD-ve after 4 courses of FCR, suggesting that some patients may not require 6 courses of therapy. Myelotoxicity remains an issue with 7 patients not completing therapy. Longer follow-up will clarify whether shortened therapy will have an impact on MRD+ve reversion, time to re-treatment and survival. Disclosures: No relevant conflicts of interest to declare.

Assessment Of Minimal Residual Disease In Acute Myeloblastic Leukemia In Multiparameter Flow Cytometry

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2613-2613 ◽  
Author(s):  
Francis Lacombe ◽  
Kaoutar Allou ◽  
Christine Arnoulet ◽  
Lydia Campos ◽  
Adrienne de Labarthe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Internal Control ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Molecular Techniques ◽  
Multiparameter Flow Cytometry ◽  
Minimal Residual

Abstract Acute myeloid leukemias (AML) represent a vast and complex group of diseases where numerous molecular lesions have been and keep being described. From the immunophenotypic point of view, probably because of the variety of cells in the myeloid lineage, a rather large variety also exists. The detection of minimal residual disease (MRD) in such conditions therefore meets the challenge of tracking the proper anomaly and correctly separate leukemic cells from their normal counterparts. In an oligocentric project initiated in France in late 2006, ten cytometry platforms and six molecular biology laboratories collaborated to detect MRD concomitantly in flow and with molecular techniques. The flow cytometry part of this work is reported here. A total of 307 consecutive patients were tested at diagnosis with a comprehensive common panel allowing for the detection of immature markers and potential leukemia-associated immunophenotypic patterns. Follow-up samples were planned to be obtained after induction and at the end of treatment, with an optional control before the second consolidation. In fine, 274 patients had at least one point of follow up. All samples were tested in a technique of whole bone marrow lysis no wash, avoiding any cell loss. The flow cytometry panel comprised ten five-colors tubes, all containing CD45 as gating marker. A newly developed strategy was devised to analyse MRD data. The approach of the GTLLF (Arnoulet, Cytometry part B, 2011) was first applied to properly identify mature polymorphonuclears, monocytes and lymphocytes, allowing for a negative Boolean selection of immature cells in the region dubbed “bermudes” by this group. Focusing on this area, each combination of the four markers tested together with CD45 was then displayed in a total of six biparametric histograms. For each of them, on the diagnosis sample, quadrant gates were constructed so that the lower left one contained no blast cells. A Boolean operation was then designed to exclude all these six areas, thereby combining the positive blasts present in the three other parts of each quadrant. The resulting population was visualized on a CD45/side scatter biparametric histogram to check that the cells appeared as a focused cluster at a precise position. The same strategy was then applied for each patient’s consecutive samples, always checking whether any cells identified with this protocol displayed the scattered pattern of cells engaged in maturation (no MRD) or constituted a focused population without maturation (positive MRD). The amount of MRD was then calculated taking into account as denominator the whole population of nucleated cells in the sample (excluding debris on a live gate). As internal control a specific feature of the Kaluza software was used to merge samples obtained for a given patient in order to display on the same worksheet the diagnosis and follow up samples using the principal component separation provided by the “radar” tool of this software. This original method proved to be easily applicable and provide a consistent help for MRD interpretation. All patients could be assessed for MRD with only two of the ten tubes used. These contained the following combinations : CD15, CD13, CD33, CD34, CD45 and CD7, CD117, CD33, CD34, Cd45. At diagnosis, any combination of expression of CD13, CD33, CD117 and CD34 could be observed, the percentage of positive cases for each of these antigens being respectively 86%, 89%, 81% and 58%. As a whole, 93% of the follow-up samples (MRD) tested contained less than 5% of cells with an immunophenotype comparable to that of diagnosis. This figure was 77% for less than 1% and 43% for less than 0.1%. The strategy devised for files analysis was easily applicable for all patients except those with myelomonocytic leukemia. For some of them, separation of the blasts from the monocytic compartment could be problematic in regenerating bone marrow samples. In conclusion, the flow cytometry part of this multicenter study allowed to establish that the combination of CD45 with CD13, CD33, CD117 and CD34 with the additional information provided by CD5 and CD7 represents a quasi-universal panel, now easy to implement on instruments with 8 or 10 detectors, for the detection of MRD in multiparameter in flow cytometry. Moreover, a powerful strategy of listmodes analysis was developed allowing for the direct comparison of several samples from the same patients and/or of a given sample and normal (control) bone marrow. Disclosures: No relevant conflicts of interest to declare.

Depth of response and minimal residual disease status in ultra high-risk multiple myeloma and plasma cell leukemia treated with daratumumab, bortezomib, lenalidomide, cyclophosphamide and dexamethasone (Dara-CVRd): Results of the UK optimum/MUKnine trial.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 8001-8001
Author(s):  
Martin F. Kaiser ◽  
Andrew Hall ◽  
Katrina Walker ◽  
Ruth De Tute ◽  
Sadie Roberts ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Plasma Cell ◽  
Residual Disease ◽  
Cell Leukemia ◽  
Ultra High Risk ◽  
Grade 3 ◽  
Minimal Residual

8001 Background: Patients with ultra high-risk (UHiR) newly diagnosed multiple myeloma (NDMM) and patients with plasma cell leukemia (PCL) continue to have dismal outcomes and are underrepresented in clinical trials. Recently, improved responses with anti-CD38 monoclonal antibody combination therapy have been reported for NDMM patients. We report here outcomes for NDMM UHiR and PCL patients treated in the OPTIMUM/MUKnine (NCT03188172) trial with daratumumab, cyclophosphamide, bortezomib, lenalidomide, dexamethasone (Dara-CVRd) induction, augmented high-dose melphalan (HDMEL) and ASCT. With final analysis follow-up surpassed in Feb 2021, we report here early protocol defined endpoints from induction to day 100 post ASCT. Methods: Between Sep 2017 and Jul 2019, 107 patients with UHiR NDMM by central trial genetic (≥2 high risk lesions: t(4;14), t(14;16), t(14;20), gain(1q), del(1p), del(17p)) or gene expression SKY92 (SkylineDx) profiling, or with PCL (circulating plasmablasts > 20%) were included in OPTIMUM across 39 UK hospitals. Patients received up to 6 cycles of Dara-CVRd induction, HDMEL and ASCT augmented with bortezomib, followed by Dara-VR(d) consolidation for 18 cycles and Dara-R maintenance. Primary trial endpoints are minimal residual disease (MRD) status post ASCT and progression-free survival. Secondary endpoints include response, safety and quality of life. Data is complete but subject to further data cleaning prior to conference. Results: Median follow-up for the 107 patients in the safety population was 22.2 months (95% CI: 20.6 – 23.9). Two patients died during induction due to infection. Bone marrow aspirates suitable for MRD assessment by flow cytometry (10-5 sensitivity) were available for 81% of patients at end of induction and 78% at D100 post ASCT. Responses in the intention to treat population at end of induction were 94% ORR with 22% CR, 58% VGPR, 15% PR, 1% PD, 5% timepoint not reached (TNR; withdrew, became ineligible or died) and at D100 post ASCT 83% ORR with 47% CR, 32% VGPR, 5% PR, 7% PD, 10% TNR. MRD status was 41% MRDneg, 40% MRDpos and 19% not evaluable post induction and 64% MRDneg, 14% MRDpos and 22% not evaluable at D100 post ASCT. Responses at D100 post ASCT were lower in PCL with 22% CR, 22% VGPR, 22% PR, 22% PD, 12% TNR. Most frequent grade 3/4 AEs during induction were neutropenia (21%), thrombocytopenia (12%) and infection (12%). Grade 3 neuropathy rate was 3.7%. Conclusions: This is to our knowledge the first report on a trial for UHiR NDMM and PCL investigating Dara-CVRd induction and augmented ASCT. Response rates were high in this difficult-to-treat patient population, with toxicity comparable to other induction regimens. However, some early progressions highlight the need for innovative approaches to UHiR NDMM. Clinical trial information: NCT03188172.

Moving Beyond Autologous Transplantation in Multiple Myeloma: Consolidation, Maintenance, Allogeneic Transplant, and Immune Therapy

2016 ◽  
pp. 210-221 ◽  
Author(s):  
Amrita Krishnan ◽  
Ravi Vij ◽  
Jesse Keller ◽  
Binod Dhakal ◽  
Parameswaran Hari
Keyword(s):  
Multiple Myeloma ◽  
Disease Control ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
The Other ◽  
Novel Agents ◽  
Patient Specific ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Minimal Residual

For multiple myeloma, introduction of novel agents as part of the front-line treatment followed by high-dose chemotherapy and autologous hematopoietic stem cell transplantation (ASCT) induces deep responses in a majority of patients with this disease. However, disease relapse is inevitable, and, with each relapse, the remission duration becomes shorter, ultimately leading to a refractory disease. Consolidation and maintenance strategy after ASCT is one route to provide sustained disease control and prevent repeated relapses. Though the consolidation strategy remains largely confined to clinical trials, significant data support the efficacy of consolidation in improving the depth of response and outcomes. There are also increasing rates of minimal residual disease–negativity with additional consolidation therapy. On the other hand, maintenance with novel agents post-transplant is well established and has been shown to improve both progression-free and overall survival. Evolving paradigms in maintenance include the use of newer proteasome inhibitors, immunotherapy maintenance, and patient-specific maintenance—a concept that utilizes minimal residual disease as the primary driver of decisions regarding starting or continuing maintenance therapy. The other approach to overcome residual disease is immune therapeutic strategies. The demonstration of myeloma-specific alloimmunity from allogeneic transplantation is well established. More sophisticated and promising immune approaches include adoptive cellular therapies, tumor vaccines, and immune checkpoint manipulations. In the future, personalized minimal residual disease–driven treatment strategies following ASCT will help overcome the residual disease, restore multiple myeloma–specific immunity, and achieve sustained disease control while minimizing the risk of overtreatment.

scholarly journals Minimal residual disease may predict bone marrow relapse in patients with hairy cell leukemia treated with 2-chlorodeoxyadenosine

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1556-1560 ◽  
Author(s):  
S Wheaton ◽  
MS Tallman ◽  
D Hakimian ◽  
L Peterson
Keyword(s):  
Bone Marrow ◽  
Minimal Residual Disease ◽  
Hairy Cell Leukemia ◽  
Residual Disease ◽  
Hairy Cell ◽  
Cell Leukemia ◽  
Hematoxylin And Eosin ◽  
Anti Cd20 ◽  
Minimal Residual

Minimal residual disease (MRD) can be detected in bone marrow core biopsies of patients with hairy cell leukemia (HCL) after treatment with 2-chlorodeoxyadenosine (2-CdA) using immunohistochemical (IHC) techniques. The purpose of this study was to determine whether the presence of MRD predicts bone marrow relapse. We studied paraffin- embedded bone marrow core biopsies from 39 patients with HCL in complete remission (CR) 3 months after a single cycle of 2-CdA. Biopsies performed 3 months posttherapy and annually thereafter were examined by routine hematoxylin and eosin (H&E) staining and IHC using the monoclonal antibodies (MoAbs) anti-CD45RO, anti-CD20, and DBA.44. At 3 months after therapy, 5 of 39 (13%) patients had MRD detectable by IHC that was not evident by routine H&E staining. Two of the five patients (40%) with MRD at 3 months have relapsed, whereas only 2 of 27 (7%) patients with no MRD and at least 1 year of follow up relapsed (P = .11). Over the 3-year follow-up period, two additional patients developed MRD. Overall, three of six (50%) patients with MRD detected at any time after therapy have relapsed, whereas only 1 of 25 (4%) patients without MRD has relapsed (P = .016). These data suggest that the presence of MRD after treatment with 2-CdA may predict relapse.

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.

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