FLT3-ITD measurable residual disease from the QuANTUM-First trial

Next-Generation Sequencing (NGS)-Based Measurable Residual Disease (MRD) Monitoring in Acute Myeloid Leukemia with FLT3 Internal Tandem Duplication (FLT3-ITD+ AML) Treated with Additional Midostaurin

FLT3/ITD-driven noncanonical STAT1 S727 phosphorylation upregulates CD276, mediating CD8positive- T cell exhaustion and immune evasion in AML

Difference in FLT3 Mutational Status between Diagnosis and Relapsed/Refractory Acute Myeloid Leukemia: A Meta-Analysis

MRD Monitoring of Non-DTA Genes By Digital Droplet PCR in the Early Period after Allogeneic Hematopoietic Stem Cell Transplantation Can Effectively Predict Patients at High Risk of Relapse

Ultra-Sensitive Assessment of Measurable Residual Disease (MRD) in Peripheral Blood (PB) of Multiple Myeloma (MM) Patients Using Bloodflow

Prognostic Role of Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in Acute Myeloid Leukemia Patients with FMS3-like Tyrosine Kinase-3 Internal Tandem Duplication (FLT3-ITD)

Nucleophosmin‐1 (NPM1) mutations in acute myeloid leukemia (AML) confer a survival advantage in the absence of FLT3‐internal tandem duplication (FLT3‐ITD). Here, we investigated the main predictors of outcome after allogeneic hematopoietic stem cell transplantation (allo‐HCT). We identified 1572 adult (age ≥ 18 year) patients with NPM1‐mutated AML in first complete remission (CR1:78%) or second complete remission (CR2:22%) who were transplanted from matched sibling donors (30.8%) or unrelated donors (57.4%) between 2007 and 2019 at EBMT participating centers. Median follow‐up for survivors was 23.7 months. FLT3‐ITD was present in 69.3% of patients and 39.2% had detectable minimal/measurable residual disease (MRD) at transplant. In multivariate analysis, relapse incidence (RI) and leukemia‐free survival (LFS) were negatively affected by concomitant FLT3‐ITD mutation (HR 1.66 p = 0.0001, and HR 1.53, p < 0.0001, respectively), MRD positivity at transplant (HR 2.18, p < 10−5 and HR 1.71, p < 10−5, respectively), and transplant in CR2 (HR 1.36, p = 0.026, and HR 1.26, p = 0.033, respectively), but positively affected by Karnofsky score ≥90 (HR 0.74, p = 0.012, and HR 0.7, p = 0.0002, respectively). Overall survival (OS) was also negatively influenced by concomitant FLT3‐ITD (HR 1.6, p = 0.0001), MRD positivity at transplant (HR 1.61, p < 10−5), and older age (HR 1.22 per 10 years, p < 0.0001), but positively affected by matched sibling donor (unrelated donor: HR 1.35, p = 0.012; haploidentical donor: HR 1.45, p = 0.037) and Karnofsky score ≥90 (HR 0.73, p = 0.004). These results highlight the independent and significant role of FLT3‐ITD, MRD status, and disease status on posttransplant outcomes in patients with NPM1‐mutated AML allowing physicians to identify patients at risk of relapse who may benefit from posttransplant prophylactic interventions.

Total Marrow and Lymphoid Irradiation (TMLI) -Based Conditioning Regimen in Patients with Acute Leukemia Undergoing Allogeneic Hematopoietic Cell Transplant with Measurable Residual Disease

Here we describe reference materials for measurable residual disease (MRD) assay design and validation as well as initial data from testing. The analytical validation of liquid biopsy-based assays that attempt to monitor for the disappearance and reemergence of cancer can be challenging due to the need for reference materials that allow for the assessment of sensitivity and specificity at variant allele frequencies (VAFs) that can be over an order of magnitude below those that can be detected reliably by typical circulating tumor DNA (ctDNA) assays. At such low VAFs, a given plasma sample may only contain limited somatic variants - and only at a few copies - which is why some MRD assays focus on monitoring only patient-specific somatic variants and do not analyze other parts of the genome. However, this also means that validation should include the steps needed to monitor patient-specific somatic variants. Therefore, we designed our reference materials for a tumor/normal workflow as a set of three components. First, one cell line is a source of normal DNA that can be used to assess specificity. Second, a germline SNP-matched cell line provides hundreds to thousands of additional somatic variants. Third, blends of fragmented and sized DNA are used to mimic circulating cell-free DNA (ccfDNA) and serve as the input for MRD assays at VAFs from 0.1% to 0.01% (one mutant copy per 3.5 to 35 ng of ccfDNA) and at 0%. As a proof of concept, we analyzed three cell lines for somatic variants by Whole Exome Sequencing (WES). Using the resulting data, digital PCR assays were designed and two different error corrected NGS assays were customized to measure some of the identified variants. Based on past experiments, A&amp;gt;G (T&amp;gt;C) and C&amp;gt;T (G&amp;gt;A) changes had the highest background in NGS data, so these were generally avoided. The tumor and normal samples were used to assess the performance of a given customized assay to measure a variant. Digital PCR was used to verify VAFs in the ccfDNA format, which proved challenging for 0.01% given the expectation of approximately one positive droplet per assay well. NGS was then used to look for the variants using around 50 ng of input per reaction. While the detection of 0.1% VAFs appeared to be feasible with customized off-the-shelf assays, 0.01% was difficult given the need to measure a variant-containing region over 10,000 times to observe a somatic variant - if it was included in the library - and to measure each starting molecule around 10 times for error correction, which required a depth of greater than 100,000 for 20 of several hundred identified candidate variants. While we did not evaluate the reference materials with emerging clinical MRD assays and focused our initial testing on customized off-the-shelf assays, these reference materials should enable the design, validation, and evaluation of MRD assays. Citation Format: Yves Konigshofer, Matthew G. Butler, Jessica Dickens, Omoshile Clement, Andrew Anfora, Dan Brudzewsky, Bharathi Anekella, Russell K. Garlick. Reference materials for measurable residual disease (MRD) monitoring [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1978.

Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. To increase detection sensitivity of FLT3-ITD, we developed new detection algorithm using next generation sequencing (NGS) data. We validated results using nested polymerase chain reaction (PCR) methods. We compared results of NGS data, nested PCR and conventional PCR methods. First, using whole exome sequencing data of 83 AML patients, we applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, we deigned two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was deigned to target previously reported regions which are frequently occurred ITD mutation. PCR reactions of two steps were performed using the PCR primers sequentially. In these 83 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, this resulted in positive FLT3-ITD in 11 patients (11/83, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all of 11 patients. Nested PCR detected additional 4 patient positive for FLT3-ITD in this population. For 68 patients, FLT3-ITD was negative by both NGS and nested PCR method. Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population. And the concordance rate of NGS method and nested PCR was 95.2% (79/83). Then we investigated clinical significance of sensitive FLT3-ITD detection. For this, we performed nested PCR and conventional PCR at the same time in 238 AML patients to detect FLT3-ITD. Positive rate for FLT3-ITD was 20% (48/238) and 10% (24/238) by nested PCR and conventional PCR respectively. When survival analysis was performed, among patients with negative FLT3-ITD result by conventional PCR, patients who showed positive for FLT3-ITD by nested PCR had shorter overall survival compared to those who showed negative for FLT3-ITD by nested PCR. (p = 0.03). This implies that sensitive FLT3-ITD detection using nested PCR is clinically meaningful. Diagnosis of FLT3-ITD is very important genetic factor, leading a therapeutic direction for AML patient. Here we report that we have developed alternative more sensitive detection methods for FLT3-ITD based on nested PCR and NGS. Sensitive detection of FLT3-ITD was clinically meaningful, suggesting that these methods should be incorporated in a future clinical practice. Also, we want to note that, NGS method is capable of quantifying FLT3-ITD size and amount in AML patients. Citation Format: Daeyoon Kim, Yoojin Hong, Youngil Koh, Sung-Soo Yoon, Choong-Hyun Sun, Kwang-Sung Ahn, Seungmook Lee, Hongseok Yun, Suyeon Lee. Improved sensitive detection method of FLT3 (FMS-like tyrosine kinase) internal tandem duplication (ITD) mutation using next-generation sequencing technology and nested PCR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3186.

Study Of Activity Of E6201, a Dual FLT3 and MEK Inhibitor, In Acute Myelogenous Leukemia With FLT3 Or RAS Mutation

Patient Specific Droplet Digital PCR Targeting FLT3-ITD Allows to Detect Small Clones in Acute Leukemias at Diagnosis and during Follow-up

Outcome of Allogeneic Stem Cell Transplantation in FLT3-TKD-Mutated AML - a Study on Behalf of the Acute Leukemia Working Party of the EBMT

Ponatinib in patients with refractory acute myeloid leukaemia: findings from a phase 1 study

Measurable residual disease (MRD) has progressively taken a central role in the field of hematological malignancies, not only as a reliable marker of quality of response to treatment, but also as a guide for the decision-making therapeutic choice. Accordingly, MRD is increasingly incorporated in experimental trials and in daily clinical practice, potentially representing a surrogate biomarker to accelerate drug development and approval. In chronic [Benintende G. et al, https://doi.org/10.3389/fonc.2023.1112616; Robak T. et al, https://doi.org/10.3389/fonc.2022.976374] and acute [Tettero JM. Et al, https://doi.org/10.3389/fonc.2022.999822] blood malignancies, either in the adult or in the pediatric [Lee JW. Et al, https://doi.org/10.3389/fonc.2022.957743] setting, MRD can be considered a reliable prognostic biomarker, in that it can provide an estimate of clinical response [Visentin A. et al, https://doi.org/10.3389/fonc.2022.1033413]. This information may be particularly relevant in those hematological malignancies that are inherently characterized by a high risk of recurrence, such as acute leukemias [Chiusolo P. et al, https://doi.org/10.3389/fonc.2022.994366; Malagola M. et al, https://doi.org/10.3389/fonc.2023.1133418]. In this subset, MRD might be incorporated into clinical trials as a “therapeutic target” to reduce disease burden before curative-intended strategies (including hematopoietic stem cells transplantation) or to indicate treatment de-intensification to spare unnecessary toxicities in patients with no evidence of residual disease [Tecchio C. et al, https://doi.org/10.3389/fonc.2023.1047554]. Potential consequence of this assessment may eventually improve the clinical outcome and also have a favorable impact on the financial cost of the overall treatment strategy. Indeed, the probability of hospitalizing patients achieving MRD negativity is usually lower as shorter is their inpatient stay.Besides giving a reliable estimate of the quality of response, MRD monitoring after treatment may also allow to significantly shorten the time to new drug approval if validated as surrogate endpoint for overall and disease-free survival. Based on this, the U.S Food & Drug Administration (FDA) has recently released a guidance document for the use of MRD in clinical trials testing new drugs for approval. According to this document, the assumption that MRD negativity correlates with a relatively small amount of residual cancer cells, thus representing a “biologically plausible” surrogate for a longer survival, should be actively pursued in clinical trials. Therefore, several trials are now incorporating MRD as an endpoint to accelerate new drug testing and approval, particularly in Acute and Chronic Lymphocytic Leukemia and Multiple Myeloma.From the technical standpoint, the innate heterogeneous nature of hematological malignancies has prompted the improvement of sensitivity and specificity of the available techniques, but also the design of new tools to track cancer populations more efficiently than “standard” MRD might do. This may be particularly relevant for the identification of cancer stem cells, which are thought to be responsible for disease relapse in those cases of apparent MRD negativity, or the refinement of post-transplant chimerism assessment to identify an impending relapse. Similarly, there is accumulating evidence on the role of Next Generation Sequencing (NGS)- and Digital-Polymerase-Chain-Reaction (PCR)-based techniques for MRD determination beside Reverse-Transcriptase quantitative PCR and Multiparametric Flow-Cytometry [Pacelli P. et al, https://doi.org/10.3389/fonc.2022.1057713; Assanto GM. Et al, https://doi.org/10.3389/fonc.2023.1152467]. In which type of disease these novelties will become stand-alone techniques is not known yet. Irrespective of the clinical subset and of the source to be tested for MRD (peripheral blood rather than bone marrow or other tissues), assessing the quality of the sample is critical to ensure the reliability of the assay. This can be particularly critical when testing the bone marrow, since either the background noise due to normal hematopoiesis or poor quality of samples (e.g. hemodilution) can significantly reduce the sensitivity and specificity of the tests [Vigliotta I. et al, https://doi.org/10.3389/fonc.2022.1001048]. However, since no consensus has been established yet on criteria for samples’ quality acceptability, specific guidelines to address this issue are needed in the near future. In conclusion, thanks to the accumulating evidences from prospective and retrospective MRD-centered trials, “why” testing MRD (e.g. development of MRD driven strategies) is becoming progressively clear. Nevertheless, until harmonization-standardization efforts will not be accomplished by the scientific community, “who” (e.g. patients that may benefit from testing), “what” (e.g. which biomarkers are suitable for MRD monitoring), where (e.g. which is the optimal source for MRD monitoring), when (e.g. which timepoints are crucial for clinical decision-making) and “how” (e.g. which technique is fit for which patient) to assess MRD still remain open questions (figure 1).