Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry‐based leukemic cell enrichment followed by mutational profiling

Abstract Abstract 1470 Investigation of minimal residual disease (MRD) after remission induction (RI) therapy provides important information for risk assessment in patients with acute myeloid leukemia (AML). The presence of immature and chemotherapy-resistant leukemic stem cells (LSC) within the bulk of AML blasts at diagnosis and in post-induction bone marrow (BM) may lead to relapses. Nevertheless, whether the frequency of cells with LSC characteristics and their clearance after induction is correlated with prognosis has not been established. Using four-color multiparametric flow cytometry (MFC), BM quantification of leukemic associated phenotypes (LAPs) and LSCs was performed at diagnosis and after the first RI from 37 AML patients, excluding acute promyelocytic leukemia, with a median age of 48 years and a male/female ratio of 0.95. All patients received the conventional daunorrubicin and cytarabin (3 + 7) RI chemotherapy. Complete remission (CR) was defined as BM blast count inferior to 5%. Thirty-three patients were classified according to the European Leukemia Net recommendation and 9/32 (28.1%) patients were allocated in favorable, 14/32 (43.8%) in intermediate and 9/32 (28.1%) in poor cytogenetic/molecular risk group. FLT3-ITD mutation was detected in 9/33 (27.3%) and 5/28 (17.9%) carried NPM1 mutation. MRD identification was performed on 20 erythrocyte-lysed whole BM samples after staining with a panel of directly conjugated monoclonal antibodies. Five patients were excluded from this analysis because a LAP could not be identified. Blasts gating was performed considering the low expression of CD45 and sideward scatter (SSC) and CD34 expression (these latter, when more than 20% of blasts were detected at diagnosis). Within this population, the following LAPs were investigated: CD15/CD117, HLA-DR/CD13, HLA-DR/CD33, CD2/CD56, CD19/CD11b, CD42a/CD33, CD64/CD11c or CD14/CD11c. LSCs were selected by the same CD45dim × SSC gating strategy and defined as CD34+/CD38−/CD123+. After staining procedures, at least one hundred thousand events were acquired in a FACScalibur flow cytometer and analysis was performed using the Cell Quest software. LAP and LSC quantification, at diagnosis and at days 21 to 30 of RI, was analyzed as a categorical variable defined as lower or higher than the median and was compared to the following variables: age (< or > 60 years old); WBC count (< or > 30 × 103); cytogenetic/molecular risk; and morphological CR. The comparison between LAP and LSC quantification at both time points was also assessed. Comparison of categorical variables was performed using Fisher's exact test or Yates' corrected chi-square for two or more variables, respectively. Statistical analyses were performed using SPSS 13.0 software and P < 0.05 was considered to be significant. LSC quantification at diagnosis was found at varying frequencies across different cytogenetic/molecular risk groups, being higher at the poor risk group (P = 0.041). Of note, 100% of the poor risk patients had high levels of LSC at diagnosis. In addition, the presence of FLT3-ITD mutation was associated with higher amounts of the LSC population at diagnosis (P = 0.043). The most frequent detected LAP was CD45dim/CD34+/HLA-DR+/CD13+. Low or high expression of the LAP was not correlated with the prognostic variables at diagnosis. CR rate was 83.33% and was not different in the groups with high or low levels of LSC at diagnosis. However, LAP and LSC quantification after RI were found to be correlated (P = 0.018), suggesting that the LSC subpopulation can be useful for MRD monitoring at this early treatment time-point. Therefore, LSC quantification by MFC at diagnosis can identify patients at high risk of relapse and offers the opportunity to study the stem cell compartment after chemotherapy. These findings are particularly important for the intermediate normal karyotype risk group patients, who frequently do not have specific molecular targets for MRD monitoring. Disclosures: No relevant conflicts of interest to declare.

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A Combined Score of Minimal Residual Disease (MRD) Assessment by Flow Cytometry, Cytogenetic and Molecular Markers As Well As Age Predicts Outcome and Can Potentially Guide MRD-Based Therapy in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.934.934 ◽

2011 ◽

Vol 118 (21) ◽

pp. 934-934 ◽

Cited By ~ 1

Author(s):

Thomas Köhnke ◽

Daniela Sauter ◽

Katharina Ringel ◽

Jan Braess ◽

Wolfgang Hiddemann ◽

...

Keyword(s):

Flow Cytometry ◽

Overall Survival ◽

Acute Myeloid Leukemia ◽

Induction Chemotherapy ◽

Myeloid Leukemia ◽

Residual Disease ◽

Prognostic Score ◽

Therapeutic Decisions ◽

Minimal Residual ◽

Acute Myeloid

Abstract Abstract 934 Background: Induction chemotherapy in acute myeloid leukemia (AML) has been shown to successfully induce complete remission in over 70% of patients. However, a majority of patients experience subsequent relapse. Assessment of minimal residual disease (MRD) by flow cytometry at time of aplasia, after induction and after consolidation therapy has been shown to be of prognostic relevance for relapse free survival (RFS) and overall survival (OS). However, studies utilizing MRD diagnostics to guide therapeutic decisions in adult AML (excluding APL) are yet to be performed. Methods: From the database at the Laboratory of Leukemia Diagnostics at our clinic datasets of 583 patients with newly diagnosed AML treated between 2000 and 2011 were analyzed. Patients with biphenotypic acute leukemia, M3 according to FAB classification, as well as those not treated with intensive induction chemotherapy were excluded. To be eligible for further analysis, at least two samples of bone marrow blood (at primary diagnosis and at one further timepoint during or after treatment) had to be available for MRD assessment by 3-color-flow cytometry at our laboratory. Cytogenetic and molecular risk stratification was performed at our clinic and assigned in accordance to the European LeukemiaNet (ELN) guidelines. We used Cox Proportional Hazards Regression to determine prognostic factors for OS and RFS and Kaplan-Meier estimator to determine OS and RFS of the proposed score. Results: Data of 217 Patients fulfilled the inclusion criteria and were therefore eligible for further analysis. 171 (78,8%) patients achieved CR after induction therapy. Of these patients, 120 had flow cytometry data available at time of aplasia and were included in further analysis. The median age was 54,5 y and the median OS 1007 days. Here, only “favorable” ELN risk stratification was associated with significantly longer OS (favorable vs. intermediate-I, Intermediate-II & adverse, Hazard Ratio, HR 0,36, 95% CI 0,19–0,69, p=0,0019), whereas RFS did not yield a significant difference (HR 0,64, 0,37-1,13, p=0,125). Age > 60y was associated with significantly shorter OS (HR 2,07, 1,23-3,47, p=0,0058) and RFS (HR 1,83, 1,11-3,01, p=0,018). And though leukemia-associated phenotypes (LAIP) ≥0,15% at time of aplasia were not predictive of OS (HR 1,32, 0,79–2,23, p=0,293) they were highly predictive of shorter RFS (HR 2,15, 1,30–3,55, p=0,003). Combining these three factors in a simple prognostic score (ELN risk group “favorable” = 0 points, “intermediate-I”, “intermediate-II” or “adverse” = 1 point; age > 60y = 1 point; LAIP at time of aplasia ≥0,15% = 1 point, see table I) identified three distinct groups (0 points: good, 1 point: intermediate, 2–3 points: poor, see table II) which were predictive of both OS and RFS (see figures 1 and 2). Interestingly, this score was capable of identifying a small group of patients with a very good prognosis (n=18, median OS and RFS not reached after >6 years) while at the same time equally dividing up the remaining patients within the intermediate and poor prognosis group (n=52 vs. 50, median OS 1182 vs. 677 days, median RFS 1180 vs. 334 days). Conclusion: MRD based therapeutic decisions and risk-adapted therapy have long been suggested in management of adult AML. Here, we propose a prognostic score for patients with AML achieving CR under intensive induction chemotherapy. The addition of MRD Flow to established genetic prognostic markers as well as age improves the prediction of relapse free and overall survival. Application of this score in therapeutic decisions could assist the treating physician and avoid over-treatment. To further evaluate our proposed prognostic score, it has to be applied in a prospective study for further evaluation and determination of its clinical significance. These data will be the basis for therapeutic trials guided by MRD assessment. Disclosures: No relevant conflicts of interest to declare.

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