scholarly journals Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

10.3791/56386 ◽  
2018 ◽  
Author(s):  
Jacqueline Cloos ◽  
Jeffrey R. Harris ◽  
Jeroen J.W.M. Janssen ◽  
Angele Kelder ◽  
F. Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Leukemic Stem Cells ◽  
Measurable Residual Disease ◽  
Acute Myeloid

Molecular Measurable Residual Disease Detection in Acute Myeloid Leukemia Using Error Corrected Next Generation Sequencing

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 5-6
Author(s):  
Nikhil Patkar ◽  
Anam Fatima Shaikh ◽  
Chinmayee Kakirde ◽  
Rakhi Salve ◽  
Prasanna Bhanshe ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Next Generation Sequencing ◽  
Myeloid Leukemia ◽  
Clinical Utility ◽  
Residual Disease ◽  
Time Points ◽  
Generation Sequencing ◽  
Measurable Residual Disease ◽  
Acute Myeloid

Introduction The monitoring of a patient's response to chemotherapy, called, measurable residual disease (MRD) is one of the most important predictors of outcome in Acute Myeloid Leukemia (AML). Although universally applicable, FCM-MRD for AML suffers from low sensitivity as compared to precursor B lineage acute lymphoblastic leukemia. Here, we evaluated the clinical utility of error corrected next generation sequencing (NGS) to detect MRD (NGS-MRD) in AML using single molecule molecular inversion probes (smMIPS). We compare NGS-MRD and FCM-MRD and determine their impact on patient outcome. We demonstrate that error corrected NGS-MRD at early timepoints in therapy is an independent and significant predictor of outcome in patients of AML treated with conventional therapies. Methods We created a 35 gene "hotspot" panel comprising of a pool of 302 smMIPS. In brief, this panel covers regions of 35 commonly mutated genes in AML.FLT3-ITD were detected using a novel one-step PCR based NGS assay. Post mapping, singleton reads (originating from one UMI) were discarded and consensus family based variant calling was performed. We then created a site and mutation specific error model to ascertain the relevance of an observed variant at each site. A limit of detection (LOD) experiment demonstrated a lower detection limit of 0.05%. For FLT3-ITD the LOD was 0.002%. A total of 393 adult patients of AMLwere accrued over a period of six years.Patients were treated with standard 3+7 induction followed by 3 doses of HiDAC. Allogeneic bone marrow transplantation was offered where feasible. Somatic mutations at diagnosis were evaluated using a smMIPS based 50 gene myeloid panel which was applicable to 327 patients [83.2% of AMLs, median 2 mutations per case (range 1 - 6 trackable mutations)].MRD assessment could be performed in 201 adult patients of AML in morphological remission (not performed in the rest because of suboptimal quality DNA at MRD time points or missing sample).Samples were sequenced on multiple S4 flow cells of a NovaSeq 6000 using 150PE chemistry.FCM-MRD was obtained from the bone marrow at end of induction (PI, n=200) and end of first consolidation cycle (PC, n=98). NGS-MRD sample also obtained at the same time points (PI, n=196& PC, n=127) from the bone marrow (n=266) or peripheral blood (n=45). Results The interaction of mutations that were trackable at diagnosis can be seen in Figure 1A. A total of 345 mutations could be detected in 196 patients (Figure 1B) with a median VAF of 1.01% [0.82% after exclusion of mutations in DNMT3A, TET2, ASXL1 (DTA) genes; (median of 2 mutations for PI and one for PC timepoint)]. The median consensus read coverage was 11,127 for the smMIPS assay, whereas for the FLT3-ITD assay it was 13,96,366.The median follow-up of the cohort was 42.3 months. The presence of NGS-MRD (70.9%) was associated with inferior overall survival (OS; p=0.001) [hazard ratio(HR)- 2.24; 95% confidence interval (CI)- 1.47 to 3.43] and relapse free survival (RFS; p=0.0002) [HR- 2.28; 95% CI- 1.58 to 3.31] at PI time point as well as PC time points [40.94% positive; OS (p=0.008)(HR- 1.92; 95% CI- 1.14 to 3.22) and RFS (p=0.004)(HR- 1.90; 95% CI- 1.18 to 3.05)].Similarly, FCM-MRD (44%) was predictive of inferior OS (p=0.0002)(HR- 2.08; 95% CI- 1.38 to 3.13)and RFS (p=0.0008)(HR- 1.81; 95% CI- 1.26 to 2.60) at PI as well as PC time points [21.4% positive, OS (p=0.04)(HR- 1.87; 95% CI- 0.89 to 3.91) and RFS (p=0.001)(HR- 2.38; 95% CI- 1.17 to 4.81)]. On multivariate analysis post induction NGS MRD emerged as the most important independent prognostic factor predictive of inferior outcome for OS [HR- 1.94; 95% CI-1.15 to 3.27; (p<0.0001)]as well asRFS[HR-2.05; 95% CI-1.30 to 3.23; (p<0.0001)].On incorporating results combining both the MRD modalities,patients that were positive by both techniqueshad a significantly inferior outcome with respect to OS (p=0.0002; HR- 4.66; 95% CI- 2.71 to 8.0)and RFS (p=0.0001; HR- 4.03; 95% CI- 2.51 to 6.47) at PI timepoint as well as PC timepoint [OS (p=0.02; HR- 3.73; 95% CI- 1.07 to 12.97) and RFS (p=0.0015; HR- 4.17; 95% CI- 1.27 to 13.7)] as compared to patients negative by both modalities (Figure 1E,F) Conclusion In conclusion, we demonstrate that error corrected panel-based sequencing is feasible for MRD monitoring in AML and may offer an advantage over existing techniques. Maximum clinical utility may be leveraged by combining FCM and NGS modalities. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Selective Targeting of Unique Metabolic Properties of Leukemic Stem Cells in Acute Myeloid Leukemia

2021 ◽  
pp. 21-24
Author(s):  
Benjamin Brakel
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Standard Treatment ◽  
Residual Disease ◽  
Leukemic Stem Cells ◽  
The Past ◽  
Selective Targeting ◽  
Metabolic Properties ◽  
Acute Myeloid

Current therapeutic options in the treatment of acute myeloid leukemia often succumb to high instances of relapse and subsequent mortality. Chemotherapy and radiotherapy have long been used as the standard treatment for this disease, remaining stagnant over the past few decades. Recently, a small self-renewing population of leukemic stem cells have been identified as drivers of cancer relapse and progression due to their increased resistance to anticancer therapeutics. This enables these cells to maintain a minimal residual disease and results in downstream differentiation, leading to relapse. Targeting these cells may lead to effective therapies that reduce relapse and mortality. Recently, the metabolic properties of leukemic stem cells have begun to be elucidated. Here, we discuss recent discoveries regarding the metabolism of leukemic stem cells and approaches to targeting their unique metabolic properties.  

Accurate Detection of Residual Leukemic Stem Cells In Remission Bone Marrow Predicts Relapse In Acute Myeloid Leukemia Patients

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 759-759 ◽  
Author(s):  
Monique Terwijn ◽  
Arjo P Rutten ◽  
Angèle Kelder ◽  
Alexander N Snel ◽  
Willemijn J Scholten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Consolidation Therapy ◽  
Leukemic Stem Cells ◽  
Prognostic Information ◽  
Induction Cycle ◽  
Cd34 Expression ◽  
Acute Myeloid

Abstract Abstract 759 Detecting leukemia initiating cells or leukemic stem cells (LSCs) in acute myeloid leukemia (AML) is a challenge. AML is a heterogeneous disease and since the first studies on LSCs, different immunophenotypic cell compartments have been reported to contain LSCs. For CD34 positive (CD34+) patients, the most important LSC compartment is most likely CD34+CD38-. However, both LSCs and normal hematopoietic stem cells (HSCs) reside in this compartment. Therefore, LSC detection requires the ability to accurately discriminate between LSCs and HSCs. Previous research showed that LSCs, in contrast to HSCs, expressed aberrant markers (AM, including lymphoid lineage markers and the LSC marker CLL-1). Although AM- cells are often presumed to be normal HSCs, AM expression can be absent or very weak on LSCs and AM- cells can therefore be malignant too. We previously described that in 30% of the patients, besides the AM expression, additional FACS parameter (forward scatter (FSC) and sideward scatter (SSC) and/or CD34 expression levels) could also be used to detect LSCs (Terwijn et al., ASH 2009 abstract 399). To prove that the CD34+CD38- fraction contains both normal and malignant SCs, and in addition, to prove that AM- cells can be malignant too, we studied molecular aberrancies (3 cases of FLT3-ITD positive AML) in the relevant cell fractions. 1: CD34+CD38-AM+ cells proved to be FLT3-ITD positive in all three cases. In addition, these cells initiated leukemic engraftment in NOD-SCID IL-2Rγ -/- mice in 2/2 cases. 2: The CD34+CD38-AM- cells with a FSC/SSC low or CD34 high character were FLT3-ITD negative in 3/3 cases and initiated multilineage engraftment in mice in 2/2 cases, indicating that these are HSCs. 3: The CD34+CD38-AM- cells with a FSC/SSC high or CD34 low phenotype were FLT3-ITD positive in 2/2 cases and therefore malignant, indicating that AM expression alone is not always sufficient in discriminating HSCs from LSCs. Since therapy surviving LSCs are thought to be responsible for relapses, we hypothesized that LSC frequency, as a fraction of total WBC, detected in remission bone marrow (BM) would provide prognostic information. Using AM as a primary gating strategy and, when applicable, FSC/SSC and/or CD34 expression, as a secondary gating step, LSC analysis was performed in 112 CD34+ AML patients (<60 years). In 76/112 patients, LSCs were detected using the most specific AM (CLL-1 (n=45), CD7 (n=19), CD11b (n=4), CD56 (n=3), CD22 (n=2), CD19 (n=2) and CD36 (n=1)) as a primary gating strategy. In 20 of these patients, a secondary gating step in FSC/SCC (n=16) and CD34 expression (n=4) refined the malignant population. In 36/112 patients, no AM was expressed on the CD34+CD38- cells, but in 25 cases, FSC/SSC could be used to gate the malignant fraction, in 6 cases combined with CD34 expression. Thus, in total, 101/112 patients were monitored for LSC frequency. After first cycle of chemotherapy, using a cut-off 5 × 10−6, patients with LSC frequency above cut-off (LSC+) showed shorter overall survival (OS, p=0.06) and especially relapse-free survival (RFS, p=0.001) as compared to LSC- patients. Median RFS was 11 months vs >36 months, respectively (fig A). Also after second induction cycle, high LSC frequency (>5 × 10−6) predicted shorter OS and RFS (both p<0.001). Median RFS of LSC+ patients was 8 months, that of LSC- patients >36 months (fig B). After consolidation therapy, 2 × 10−6 was the most optimal cut-off to define LSC+ and LSC- patients. OS and RFS were significantly shorter in LSC+ patients (p=0.037 and p=0.001, respectively), with a median RFS of 13 months vs. > 36 months (figure C). The relative risk of relapse was 5.0 (95% C.I 1.8–14.0) after first induction cycle, 4.7 (95% C.I.2.2-10.1) after second cycle and 8.5 (95% C.I 1.8– 41.4) after consolidation therapy. Multivariate analysis including WBC count, cytogenetic risk profile and the number of cycles needed to reach CR showed that LSC frequency is an independent factor for RFS (1st cycle p=0.01, 2nd cycle p<0.001, consolidation p=0.038). With this gating strategy, 90% of CD34+ AML patients could be monitored for residual LSCs, resulting in accurate prognostic information which is valuable for risk stratification-based treatment. In addition, the prospective isolation of LSCs and HSCs from diagnosis AML BM might identify more selective therapies that eradicate LSCs, while leaving HSCs intact. This work was supported by Netherlands Cancer Foundation KWF. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acute Myeloid Leukemia Measurable Residual Disease Detection by Flow Cytometry in Peripheral Blood Versus Bone Marrow

Blood ◽  
2020 ◽  
Author(s):  
Colin Douglas Godwin ◽  
Yi Zhou ◽  
Megan Othus ◽  
Mallette M. Asmuth ◽  
Carole Shaw ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Disease Detection ◽  
Measurable Residual Disease ◽  
Acute Myeloid

scholarly journals Escape From Treatment; the Different Faces of Leukemic Stem Cells and Therapy Resistance in Acute Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Noortje van Gils ◽  
Fedor Denkers ◽  
Linda Smit
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Residual Disease ◽  
Therapy Response ◽  
Therapy Resistance ◽  
Leukemia Cells ◽  
Leukemic Stem Cells ◽  
Acute Myeloid

Standard induction chemotherapy, consisting of an anthracycline and cytarabine, has been the first-line therapy for many years to treat acute myeloid leukemia (AML). Although this treatment induces complete remissions in the majority of patients, many face a relapse (adaptive resistance) or have refractory disease (primary resistance). Moreover, older patients are often unfit for cytotoxic-based treatment. AML relapse is due to the survival of therapy-resistant leukemia cells (minimal residual disease, MRD). Leukemia cells with stem cell features, named leukemic stem cells (LSCs), residing within MRD are thought to be at the origin of relapse initiation. It is increasingly recognized that leukemia “persisters” are caused by intra-leukemic heterogeneity and non-genetic factors leading to plasticity in therapy response. The BCL2 inhibitor venetoclax, combined with hypomethylating agents or low dose cytarabine, represents an important new therapy especially for older AML patients. However, often there is also a small population of AML cells refractory to venetoclax treatment. As AML MRD reflects the sum of therapy resistance mechanisms, the different faces of treatment “persisters” and LSCs might be exploited to reach an optimal therapy response and prevent the initiation of relapse. Here, we describe the different epigenetic, transcriptional, and metabolic states of therapy sensitive and resistant AML (stem) cell populations and LSCs, how these cell states are influenced by the microenvironment and affect treatment outcome of AML. Moreover, we discuss potential strategies to target dynamic treatment resistance and LSCs.

High Proportion of Leukemic Stem Cells at Diagnosis Is Correlated with Unfavorable Prognosis in Childhood Acute Myeloid Leukemia

2011 ◽  
Vol 28 (2) ◽  
pp. 91-99 ◽  
Author(s):  
Kai-Erik Witte ◽  
Jörg Ahlers ◽  
Iris Schäfer ◽  
Maya André ◽  
Gunter Kerst ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Stem Cells ◽  
Childhood Acute Myeloid Leukemia ◽  
Acute Myeloid
Sign in / Sign up

Export Citation Format

Share Document