Next-generation sequencing in the diagnosis and minimal residual disease assessment of acute myeloid leukemia.
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ABSTRACT:
SUBMITTER: Levine RL
PROVIDER: S-EPMC6518900 | biostudies-literature | 2019 May
REPOSITORIES: biostudies-literature
Publications
Next-generation sequencing in the diagnosis and minimal residual disease assessment of acute myeloid leukemia.
Levine Ross L RL Valk Peter J M PJM
Haematologica 20190328 5
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Project description:Detection of minimal residual disease predicts adverse outcome in patients with acute myeloid leukemia. Currently, minimal residual disease may be detected by RQ-PCR or flow cytometry, both of which have practical and diagnostic limitations. Here, we describe a next-generation sequencing assay for minimal residual disease detection in NPM1-mutated acute myeloid leukemia, which encompasses ∼60% of patients with normal karyotype acute myeloid leukemia. Exon 12 of NPM1 was PCR amplified using sequencing adaptor-linked primers and deep sequenced to enable detection of low-prevalence, acute myeloid leukemia-specific activating mutations. We benchmarked our results against flow cytometry, the standard of care for acute myeloid leukemia minimal residual disease diagnosis at our institution. The performance of both approaches was evaluated using defined dilutions of an NPM1 mutation-positive cell line and longitudinal clinical samples from acute myeloid leukemia patients. Using defined control material, we found this assay sensitive to approximately 0.001% mutant cells, outperforming flow cytometry by an order of magnitude. Next-generation sequencing was precise and semiquantitative over four orders of magnitude. In 22 longitudinal samples from six acute myeloid leukemia patients, next-generation sequencing detected minimal residual disease in all samples deemed negative by flow cytometry. Further, in one-third of patients, sequencing detected alternate NPM1 mutations in addition to the patient's index mutation, consistent with tumor heterogeneity. Next-generation sequencing provides information without prior knowledge of NPM1 mutation subtype or validation of allele-specific probes as required for RQ-PCR assays, and without generation and interpretation of complex multidimensional flow cytometry data. This approach may complement current technologies to enhance patient-specific clinical decision-making.
| S-EPMC4201902 | biostudies-literature
Project description:Differential induction therapy of all subtypes of acute myeloid leukemia other than acute promyelocytic leukemia is impeded by the long time required to complete complex and diverse cytogenetic and molecular genetic analyses for risk stratification or targeted treatment decisions. Here, we describe a reliable, rapid and sensitive diagnostic approach that combines karyotyping and mutational screening in a single, integrated, next-generation sequencing assay. Numerical karyotyping was performed by low coverage whole genome sequencing followed by copy number variation analysis using a novel algorithm based on in silico-generated reference karyotypes. Translocations and DNA variants were examined by targeted resequencing of fusion transcripts and mutational hotspot regions using commercially available kits and analysis pipelines. For the identification of FLT3 internal tandem duplications and KMT2A partial tandem duplications, we adapted previously described tools. In a validation cohort including 22 primary patients' samples, 9/9 numerically normal karyotypes were classified correctly and 30/31 (97%) copy number variations reported by classical cytogenetics and fluorescence in situ hybridization analysis were uncovered by our next-generation sequencing karyotyping approach. Predesigned fusion and mutation panels were validated exemplarily on leukemia cell lines and a subset of patients' samples and identified all expected genomic alterations. Finally, blinded analysis of eight additional patients' samples using our comprehensive assay accurately reproduced reference results. Therefore, calculated karyotyping by low coverage whole genome sequencing enables fast and reliable detection of numerical chromosomal changes and, in combination with panel-based fusion-and mutation screening, will greatly facilitate implementation of subtype-specific induction therapies in acute myeloid leukemia.
| S-EPMC6355503 | biostudies-literature
Project description:Measuring minimal residual disease (MRD) during treatment is valuable to identify acute lymphoblastic leukemia (ALL) patients who require intensified treatment to avert relapse. We performed the next-generation sequencing (NGS)-based immunoglobulin gene (Ig) clonality assay and evaluated its clinical implication in pediatric B-ALL patients to assess MRD. Fifty-five patients who were diagnosed and treated with de novo (n = 44) or relapsed/refractory B-ALL (n = 11) were enrolled. MRD assessment was performed using the LymphoTrack® Dx IGH and IGK assay panels. The percentage of the clonal sequences per total read count was calculated as MRD (% of B cells). The data were normalized as the proportion of total nucleated cells (TNC) by LymphoQuant™ Internal control or the B-cell proportion in each sample estimated by flow cytometry or immunohistochemistry. Clonal Ig rearrangement was identified in all patients. The normalized MRD value was significantly lower than the unnormalized MRD value (p < 0.001). When categorizing patients, 27 of 50 patients (54%) achieved normalized MRD <0.01%, while 6 of them did not achieve MRD <0.01% when applying the unnormalized value. The normalized post-induction MRD value of 0.01% proved to be a significant threshold value for both 3-year event-free survival (100% for MRD <0.01% vs. 60.9% ± 10.2% for MRD ≥0.01%, p = 0.007) and 3-year overall survival (100% for MRD <0.01% vs. 78.3% ± 8.6% for MRD ≥0.01%, p = 0.011). However, unnormalized MRD was not a significant factor for outcome in this cohort. Our study demonstrated that MRD assessment by NGS-based Ig clonality assay could be applied in most pediatric B-ALL patients. Normalized post-induction MRD <0.01% was a significant prognostic indicator.
| S-EPMC9521036 | biostudies-literature
Project description:The in-frame internal tandem duplication (ITD) of the FMS-like tyrosine kinase 3 (FLT3) gene is an important negative prognostic marker in acute myeloid leukemia (AML). FLT3-ITD monitoring is essential for patients at relapse or those receiving FLT3-targeted therapies. Fragment analysis (FA) is commonly used to detect and quantify FLT3-ITDs; however, detecting low-burden FLT3-ITDs after a treatment is challenging. We, therefore, developed a customized, next-generation sequencing (NGS)-based FLT3-ITD assay that includes a new ITD-tracing algorithm, "SEED", optimized for measurable residual disease (MRD) monitoring. NGS-SEED showed an enhanced sensitivity (0.001%) and has a superior performance over conventional fragment analysis. We further investigated the prognostic impact of MRD analyzed by NGS-SEED in AML patients who underwent allogeneic hematopoietic stem cell transplantation (HSCT). Our assay showed that the MRD assessed before and after HSCT were significantly associated with a risk of relapse and a poor overall survival, respectively, in a time-dependent analysis. Thus, this report highlighted the prognostic value of serial MRD monitoring using a sensitive method in a clinical setting of AML patients with FLT3-ITD.
| S-EPMC9776673 | biostudies-literature
Project description:Detecting persistent minimal residual disease (MRD) allows the identification of patients with an increased risk of relapse and death. In this study, we have evaluated MRD 3 months after transplantation in 106 myeloma patients using a commercial next-generation sequencing (NGS) strategy (LymphoTrack®), and compared the results with next-generation flow (NGF, EuroFlow). The use of different marrow pulls and the need of concentrating samples for NGS biased the applicability for MRD evaluation and favored NGF. Despite that, correlation between NGS and NGF was high (R2 = 0.905). The 3-year progression-free survival (PFS) rates by NGS and NGF were longer for undetectable vs. positive patients (NGS: 88.7% vs. 56.6%; NGF: 91.4% vs. 50%; p < 0.001 for both comparisons), which resulted in a 3-year overall survival (OS) advantage (NGS: 96.2% vs. 77.3%; NGF: 96.6% vs. 74.9%, p < 0.01 for both comparisons). In the Cox regression model, NGS and NGF negativity had similar results but favoring the latter in PFS (HR: 0.20, 95% CI: 0.09-0.45, p < 0.001) and OS (HR: 0.21, 95% CI: 0.06-0.75, p = 0.02). All these results reinforce the role of MRD detection by different strategies in patient prognosis and highlight the use of MRD as an endpoint for multiple myeloma treatment.
| S-EPMC7603393 | biostudies-literature
Project description:Minimal (or measurable) residual (MRD) disease provides a biomarker of response quality for which there is robust validation in the context of modern intensive treatment for younger patients with Acute Myeloid Leukemia (AML). Nevertheless, it remains a relatively unexplored area in older patients with AML. The lack of progress in this field can be attributed to two main reasons. First, physicians have a general reluctance to submitting older adults to intensive chemotherapy due to their frailty and to the unfavourable biological disease profile predicting a poor outcome following conventional chemotherapy. Second, with the increasing use of low-intensity therapies (i.e., hypomethylating agents) differing from conventional drugs in mechanism of action and dynamics of response, there has been concomitant skepticism that these schedules can produce deep hematological responses. Furthermore, age dependent differences in disease biology also contribute to uncertainty on the prognostic/predictive impact in older adults of certain genetic abnormalities including those validated for MRD monitoring in younger patients. This review examines the evidence for the role of MRD as a prognosticator in older AML, together with the possible pitfalls of MRD evaluation in older age.
| S-EPMC6070940 | biostudies-literature
Project description:BackgroundAML is a heterogeneous disease, and despite intensive therapy, recurrence is still high in AML patients who achieve the criterion for cytomorphologic remission (residual tumor burden [measurable residual disease, MRD]<5%). This study aimed to develop a targeted next-generation sequencing (NGS) panel to detect MRD in AML patients and validate its performance.MethodsWe designed an error-corrected, targeted MRD-NGS panel without using physical molecular barcodes, including 24 genes. Fifty-four bone marrow and peripheral blood samples from 23 AML patients were sequenced using the panel. The panel design was validated using reference material, and accuracy was assessed using droplet digital PCR.ResultsDilution tests showed excellent linearity and a strong correlation between expected and observed clonal frequencies (R>0.99). The test reproducibly detected MRD in three dilution series samples, with a sensitivity of 0.25% for single-nucleotide variants. More than half of samples from patients with morphologic remission after one month of chemotherapy had detectable mutations. NGS-MRD positivity for samples collected after one month of chemotherapy tended to be associated with poor overall survival and progression-free survival.ConclusionsOur highly sensitive and accurate NGS-MRD panel can be readily used to monitor most AML patients in clinical practice, including patients without gene rearrangement. In addition, this NGS-MRD panel may allow the detection of newly emerging clones during clinical relapse, leading to more reliable prognoses of AML.
| S-EPMC9989530 | biostudies-literature
Project description:We assessed minimal residual disease (MRD) detection and B-cell aplasia after tisagenlecleucel therapy for acute lymphoblastic leukemia (ALL) to define biomarkers predictive of relapse (N = 143). Next-generation sequencing (NGS) MRD detection >0 in bone marrow (BM) was highly associated with relapse. B-cell recovery [signifying loss of functional chimeric antigen receptor (CAR) T cells] within the first year of treatment was associated with a hazard ratio (HR) for relapse of 4.5 [95% confidence interval (CI), 2.03-9.97; P < 0.001]. Multivariate analysis at day 28 showed independent associations of BMNGS-MRD >0 (HR = 4.87; 95% CI, 2.18-10.8; P < 0.001) and B-cell recovery (HR = 3.33; 95% CI, 1.44-7.69; P = 0.005) with relapse. By 3 months, the BMNGS-MRD HR increased to 12 (95% CI, 2.87-50; P < 0.001), whereas B-cell recovery was not independently predictive (HR = 1.27; 95% CI, 0.33-4.79; P = 0.7). Relapses occurring with persistence of B-cell aplasia were largely CD19- (23/25: 88%). Detectable BMNGS-MRD reliably predicts risk with sufficient time to consider approaches to relapse prevention such as hematopoietic cell transplantation (HCT) or second CAR-T cell infusion. SIGNIFICANCE: Detectable disease by BMNGS-MRD with or without B-cell aplasia is highly predictive of relapse after tisagenlecleucel therapy for ALL. Clonotypic rearrangements used to follow NGS-MRD did not change after loss of CD19 or lineage switch. High-risk patients identified by these biomarkers may benefit from HCT or investigational cell therapies.See related commentary by Ghorashian and Bartram, p. 2.This article is highlighted in the In This Issue feature, p. 1.
| S-EPMC9924295 | biostudies-literature
Project description:A high proportion of patients with acute myeloid leukemia who achieve minimal residual disease negative status ultimately relapse because a fraction of pathological clones remains undetected by standard methods. We designed and validated a high-throughput sequencing method for minimal residual disease assessment of cell clonotypes with mutations of NPM1, IDH1/2 and/or FLT3-single nucleotide variants. For clinical validation, 106 follow-up samples from 63 patients in complete remission were studied by sequencing, evaluating the level of mutations detected at diagnosis. The predictive value of minimal residual disease status by sequencing, multiparameter flow cytometry, or quantitative polymerase chain reaction analysis was determined by survival analysis. The sequencing method achieved a sensitivity of 10-4 for single nucleotide variants and 10-5 for insertions/deletions and could be used in acute myeloid leukemia patients who carry any mutation (86% in our diagnostic data set). Sequencing-determined minimal residual disease positive status was associated with lower disease-free survival (hazard ratio 3.4, P=0.005) and lower overall survival (hazard ratio 4.2, P<0.001). Multivariate analysis showed that minimal residual disease positive status determined by sequencing was an independent factor associated with risk of death (hazard ratio 4.54, P=0.005) and the only independent factor conferring risk of relapse (hazard ratio 3.76, P=0.012). This sequencing-based method simplifies and standardizes minimal residual disease evaluation, with high applicability in acute myeloid leukemia. It is also an improvement upon flow cytometry- and quantitative polymerase chain reaction-based prediction of outcomes of patients with acute myeloid leukemia and could be incorporated in clinical settings and clinical trials.
| S-EPMC6355493 | biostudies-literature
Project description:Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.
| S-EPMC10435843 | biostudies-literature