Presence Of Measurable Residual Disease Research Articles

Abstract 2299: Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients

Abstract Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations. Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation. Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy. Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2299.

Utility of Immunophenotypic Measurable Residual Disease in Adult Acute Myeloid Leukemia-Real-World Context.

Introduction: One of the mainstays of chemotherapy in acute myeloid leukemia (AML) is induction with a goal to achieve morphological complete remission (CR). However, not all patients by this remission criterion achieve long-term remission and a subset relapse. This relapse is explained by the presence of measurable residual disease (MRD).Methods: We accrued 451 consecutive patients of adult AML (from March 2012 to December 2017) after informed consent. All patients received standard chemotherapy. MRD testing was done at post-induction and, if feasible, post-consolidation using 8- and later 10-color FCM. Analysis of MRD was done using a combination of difference from normal and leukemia-associated immunophenotype approaches. Conventional karyotyping and FISH were done as per standard recommendations, and patients were classified into favorable, intermediate, and poor cytogenetic risk groups. The presence of FLT3-ITD, NPM1, and CEBPA mutations was detected by a fragment length analysis-based assay.Results: As compared to Western data, our cohort of patients was younger with a median age of 35 years. There were 62 induction deaths in this cohort (13.7%), and 77 patients (17.1%) were not in morphological remission. The median follow-up was 26.0 months. Poor-risk cytogenetics and the presence of FLT3-ITD were significantly associated with inferior outcome. The presence of post-induction MRD assessment was significantly associated with adverse outcome with respect to OS (p = 0.01) as well as RFS (p = 0.004). Among established genetic subgroups, detection of MRD in intermediate cytogenetic and NPM1 mutated groups was also highly predictive of inferior outcome. On multivariate analysis, immunophenotypic MRD at the end of induction and FLT3-ITD emerged as independent prognostic factors predictive for outcome.Conclusion: This is the first data from a resource-constrained real-world setting demonstrating the utility of AML MRD as well as long-term outcome of AML. Our data is in agreement with other studies that determination of MRD is extremely important in predicting outcome. AML MRD is a very useful guide for guiding post-remission strategies in AML and should be incorporated into routine treatment algorithms.

Prognostic Impact of Blood MN1 Copy Numbers Before Allogeneic Stem Cell Transplantation in Patients With Acute Myeloid Leukemia.

High expression of the leukemia-associated gene meningioma-1 (MN1) is frequently found at diagnosis of acute myeloid leukemia (AML) and associates with adverse outcomes. The presence of measurable residual disease (MRD) in complete remission (CR) indicates high risk of relapse and worse outcome in AML patients. However, the prognostic impact of MN1 expression levels as MRD marker has not been evaluated. Digital droplet polymerase chain reaction (ddPCR) is a novel technique allowing sensitive and specific absolute gene expression quantification. We retrospectively analyzed 124 AML patients who received allogeneic hematopoietic stem cell transplantation (HSCT) in CR or CR with incomplete peripheral recovery. Absolute MN1 copy numbers in peripheral blood were assessed prior to HSCT (median 7; range 0-29 days) using ddPCR. High pre-HSCT MN1/Abelson murine leukemia viral oncogene homolog 1 gene (ABL1) copy numbers associated with a higher cumulative incidence of relapse after HSCT and-in relapsing patients-shorter time to relapse. In multivariable analysis, high pre-HSCT MN1/ABL1 copy numbers remained an independent prognosticator for relapse after HSCT. Patients with the highest pre-HSCT MN1/ABL1 copy numbers also had the highest risk of relapse. MN1 copy number assessment also added prognostic information to nucleophosmin 1 gene (NPM1) mutation- and brain and acute leukemia, cytoplasmic (BAALC) and Wilm's tumor gene 1 (WT1) expression-based MRD evaluation. Our study demonstrates the feasibility of the novel ddPCR technique for MN1/ABL1 copy number assessment as a marker for MRD. Evaluation of MN1/ABL1 copy numbers allows the identification of patients at high risk of relapse, independently of other diagnostic risk factors and MRD markers.

Development of a Novel Next-Generation Sequencing (NGS)-Based Assay for Measurable Residual Disease (MRD) in FLT3-ITD AML and Its Potential Clinical Application in Patients Treated with Chemotherapy Plus FLT3 Inhibitors

The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.Using isolated genomic DNA from bone marrow aspirates or whole-blood samples, PCR primers flanking exons 14 and 15 of the FLT3 gene were designed and highly diverse NGS libraries were generated. These libraries were then sequenced by Illumina's sequencing-by-synthesis method. The bioinformatics approach we used identifies unique FLT3-ITD mutations of varying length along with wild type sequences and calculates a mutant allelic frequency. The assay was validated using clinical samples to assess accuracy and reproducibility. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% but was capable of detecting FLT3-ITD mutations at a level as low as 0.003%.We next validated the assay using clinical samples from patients with FLT3-ITD AML. The negative prognostic impact of FLT3-ITD mutations can be mitigated in part when an FLT3 inhibitor is administered in combination with induction chemotherapy, as demonstrated in CALGB10603/RATIFY (N Engl J Med. 2017;377:454). It was reported in this study that patients treated with an FLT3 inhibitor combined with chemotherapy followed by allogeneic transplant in first remission had better overall survival than their counterparts in the control arm. One hypothesis for this outcome is that the FLT3-inhibitor-treated patients had a lower leukemic burden prior to transplant. As a pilot test of this concept, we used our MRD assay on a series of bone marrow aspirate samples collected from 10 patients with newly diagnosed FLT3-ITD AML. The patients were selected to be as uniform as possible. All patients had intermediate-risk karyotype, a detectable FLT3-ITD mutation by conventional PCR, and mutated NPM1. All patients received cytarabine-based intensive induction and achieved morphologic first remission with a single course of chemotherapy. Finally, all patients underwent allogeneic transplant in first remission. The sample analyzed for MRD was the first collected after remission induction, 5-8 weeks after the start of therapy. The investigators performing the MRD assay were blinded to the clinical data. Four patients received chemotherapy alone, while 6 were treated with chemotherapy (7+3) plus an FLT3 inhibitor. In all patients' remission samples, the MRD assay identified the FLT3-ITD mutation that precisely matched the one observed in the original diagnostic specimen. This demonstrates the sensitivity of the assay (all samples had a detectable mutation), and the unique length of each patient's mutation confers a degree of specificity not achievable with MRD detection methods that focus on other AML-associated mutations. Supporting our hypothesis was the observation that patients treated with FLT3 inhibitors had MRD levels lower than those in patients treated with chemotherapy alone (Figure).Our results help establish the role of NGS-based MRD assays for the clinical management of FLT3-ITD AML. This assay could be used to define the depth of remission, identify persistent disease, and help guide decision making in the use of FLT3 inhibitors as continuation therapy. This study provides validation of the clinical utility of our MRD assay, which will be used to analyze the remission samples from patients in the ongoing phase 3, randomized, double-blind, placebo-controlled QuANTUM-First clinical trial, in which patients with newly diagnosed FLT3-ITD AML are randomized to receive either the highly potent and selective FLT3 inhibitor quizartinib or placebo in combination with chemotherapy, followed by single-agent quizartinib as continuation therapy. [Display omitted] DisclosuresShi:Novartis: Employment, Equity Ownership; Daiichi Sankyo: Other: Provide clinical trial testing services. Chang:Daiichi Sankyo: Employment. Laing:Novartis: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Berisha:Daiichi Sankyo: Employment. Adams:Johns Hopkins University: Employment. Ding:Navigate BP: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Nakamaru:Daiichi Sankyo: Employment. Lameh:Navigate BioPharma Inc,: Employment; Daiichi Sankyo: Other: Provide clinical trial testing services. Pollner:Navigate BioPharma Inc.: Employment. Kobayashi:Daiichi Sankyo: Employment.

Pattern associated leukemia immunophenotypes and measurable disease detection in acute myeloid leukemia or myelodysplastic syndrome with mutated NPM1.

The presence of measurable residual disease after therapy is a significant risk factor of relapse in patients with acute myeloid leukemia (AML). By detecting cells with leukemia-associated immunophenotype (LAIP), multiparameter flow cytometry (MFC) can detect residual leukemia at a level significantly lower than that detected by morphology. However, changes in LAIPs during or after therapy may pose a challenge to MRD testing. AML with mutated NPM1 represents the largest subtype of AML sharing a common leukemogenic mechanism and similar LAIPs. Here, we identified a common pattern of LAIPs in myeloid blasts with mutated NPM1, and studied its stability and limit of detection after therapy. We summarized aberrancies of leukemic blasts with mutated NPM1 at diagnosis in 61 patients and paired relapse in 25 patients. In addition, we examined the detection of leukemic blasts in 590 specimens collected from 152 patients in complete remission after induction for AML/MDS-EB with mutated NPM1. Our findings demonstrate myeloid blasts with mutated NPM1 have a characteristic pattern of LAIPs that is present in nearly all cases of AML/MDS-EB with mutated NPM1 at initial diagnosis and relapse, regardless of morphologic variations, FLT3 ITD status, or karyotype abnormality. The myeloid blasts with mutated NPM1 can be detected at an approximate level of 0.1% of total leukocytes in morphologic remission with high specificity validated by clinical outcome. The characteristic pattern of LAIPs of myeloid blasts with mutated NPM1 is common and stable, and allows sensitive and specific detection of AML or MDS with mutated NPM1 after therapy. © 2018 International Clinical Cytometry Society.

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

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

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

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Prolonged gemcitabine infusion as radiosensitizer for newly diagnosed glioblastoma multiforme (GBM): A phase I study

1581 Background: The use of cytotoxic drugs concurrent with RT represents a promising approach in the combined treatment of malignant gliomas. Gemcitabine (dFdCyd) is a drug widely explored for its potential radiomimetic activity in different tumors. The present study was aimed to evaluate the dose-limiting toxicity (DLT) and maximum tolerated dose (MTD) of weekly prolonged dFdCyd infusion, administered in combination with RT as first line treatment, in adult pts affected by supratentorial GBM. Methods: Within 6 weeks after surgery, in the presence of measurable residual disease and KPS >70, pts were treated with fractionated RT at a dose of 2.0 Gy/daily fractions, 5 days a week (global dose 60 Gy). From 24 to 72 hours before the first RT application, and afterwards once weekly, pts received concurrent dFdCyd, at fixed dose rate of 10 mg/m2/min, over a total period of six weeks. Planned dose levels of dFdCyd started from 200 mg/m2/weekly (level 1), with sequential steps of 25 mg/m2/w based on toxicity. Results: Ten pts were enrolled into the study: six were male, median age 55.2 years (range 44–75), with a median KPS at baseline of 85 (SD 9.71). The median time from diagnosis to the start of treatment was six weeks (range 4–7). The median RT duration was 6 weeks (range 4–7), all but one pt received the planned dose and all pts received concomitant CT. Four pts entered at Level 1; one pt was excluded from the study, due to rapid progressive disease during the treatment. Two of the three valuable pts presented a relevant neurological worsening; on this basis the dFdCyd dose was reduced to 175 mg/m2/w (Level -1). A total of six pts were entered at Level -1, and none of them reported DLT. No hematological grade 3–4 toxicity was reported. Grade 3 non-hematological toxicity was observed in one pt (transaminases increase). After a median follow-up of 13.4 months (range 3–24), the median progression-free survival was 8 months (CI 95% 1–18), and the median overall survival was 14 months (CI 95% 12–17). Conclusions: The recommended dose of prolonged infusion of dFdCyd concomitant with RT is 175 mg/m2/w. The observed activity has been considered interesting enough to support a phase II study of this concurrent CT-RT schedule as first line treatment in GBM. [Table: see text]