NPM1-mutated Acute Myeloid Leukemia Research Articles

The Impact of DNMT3A Status on NPM1 MRD Predictive Value and Survival in Elderly AML Patients Treated Intensively.

Simple SummaryDNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of acute myeloid leukemia (AML) patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. A 4log reduction of NPM1 MRD was associated with a better outcome. DNMT3A negative patients who achieved a 4log reduction had a superior outcome to those who did not. However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identify a subgroup of patients at high risk of relapse.Minimal residual disease (MRD) is now a powerful surrogate marker to assess the response to chemotherapy in acute myeloid leukemia (AML). DNMT3A mutation has been associated with adverse outcomes. In this study, we aimed to investigate the impact of DNMT3A status on NPM1 MRD predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1-mutated AML in two French institutions were analyzed retrospectively. DNMT3A status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. Only 20.4% of FLT3-ITD patients reached ≥ 4log MRD1 reduction compared to 47.5% in FLT3wt cases. A 4log reduction of NPM1 MRD was associated with a better outcome, even in FLT3-ITD mutated patients, independent of the allelic ratio. DNMT3A negative patients who reached a 4log reduction had a superior outcome to those who did not (HR = 0.23; p < 0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and LFS in DNMT3Amut patients. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3A also identifies a subgroup of patients at high risk of relapse.

Dissecting Clonal Hematopoiesis in Tissues of Classical Hodgkin Lymphoma Patients.

Clonal hematopoiesis predisposes to hematological malignancies. However, clonal hematopoiesis is understudied in classical Hodgkin lymphoma (cHL), a mature B-cell neoplasm exhibiting the most abundant microenvironment. We analyzed clonal hematopoiesis in 40 cHL cases by sequencing microdissected tumor cells and matched normal cells from blood and/or lymph nodes. Five patients had blood and/or tissue clonal hematopoiesis. In three of five patients (all failing first-line therapy), clonal hematopoiesis spread through the tissue microenvironment extensively, and featured mutant DNMT3AR882H , KRASG60D and DNMT3AR882H +TET2Q1274 * in 33%, 92% and 60% of non-neoplastic cells, respectively. In the latter case, DNMT3A/TET2-mutant clonal hematopoiesis seeded the neoplastic clone, which was infected by the Epstein-Barr virus and showed almost no other somatic mutations exome-wide. In the former case, DNMT3A-mutant clonal hematopoiesis did not originate the neoplastic clone despite dominating the blood and B-cell lineage (~94% leukocytes; ~96% mature blood B cells), yet led to NPM1-mutated acute myeloid leukemia 6 years after therapy for cHL. Our results expand to cHL the spectrum of hematologic malignancies associated with clonal hematopoiesis.

Novel Targeted Therapeutics in Acute Myeloid Leukemia: an Embarrassment of Riches.

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow that has a poor prognosis with traditional cytotoxic chemotherapy, especially in elderly patients. In recent years, small molecule inhibitors targeting AML-associated IDH1, IDH2, and FLT3 mutations have been FDA approved. However, the majority of AML cases do not have a targetable mutation. A variety of novel agents targeting both previously untargetable mutations and general pathways in AML are currently being investigated. Herein, we review selected new targeted therapies currently in early-phase clinical investigation in AML. The DOT1L inhibitor pinometostat in KMT2A-rearranged AML, the menin inhibitors KO-539 and SYNDX-5613 in KMT2Ar and NPM1-mutated AML, and the mutant TP53 inhibitor APR-246 are examples of novel agents targeting specific mutations in AML. In addition, BET inhibitors, polo-like kinase inhibitors, and MDM2 inhibitors are promising new drug classes for AML which do not depend on the presence of a particular mutation. AML remains in incurable disease for many patients but advances in genomics, epigenetics, and drug discovery have led to the development of many potential novel therapeutic agents, many of which are being investigated in ongoing clinical trials. Additional studies will be necessary to determine how best to incorporate these novel agents into routine clinical treatment of AML.

Mutational spectrum of de novo NPM1-mutated acute myeloid leukemia patients older than 75 years

AML with mutated NPM1 occurs in all age groups. Yet, the mutational pattern is not extensively studied in the very old, which may hamper appropriate risk assessment. Herein we examined 22 cases of NPM1-mutated de novo AML in patients older than 75, with a median age of 84. All diagnostic samples were sequenced aiming for coverage of the most relevant AML-associated mutations. For comparison with younger patients, we used already published data on several cohorts. A total of 76 mutations including 50 different variants were identified in 16 recurrently mutated AML genes. Compared with younger patients, a significant enrichment of TET2 and SRSF2 was observed, together with a reduced frequency of DNMT3A mutations. Our results indicate that the mutational pattern may be different in the very old as compared to younger patients with NPM1-mutated AML. Highlights The mutational spectrum of NPM1-mutated AML in patients above 75 years displays distinct features. A significant enrichment of TET2 and SRSF2 mutations together with a reduced frequency of DNMT3A mutations was observed in the elderly. NPM1 mutation is a secondary event in the development of AML in the very old.

Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.

Flow cytometry reveals the nuances of clonal haematopoiesis

Flow cytometry reveals the nuances of clonal haematopoiesis

92 - HLA Class I Genotypes with Predicted Strong Binding Affinity to Mutated NPM1 Are Associated with Lower Relapse Risk in Matched Related or Unrelated Transplant for NPM1 Mutated Acute Myeloid Leukemia

92 - HLA Class I Genotypes with Predicted Strong Binding Affinity to Mutated NPM1 Are Associated with Lower Relapse Risk in Matched Related or Unrelated Transplant for NPM1 Mutated Acute Myeloid Leukemia

CD123 Is Consistently Expressed on NPM1-Mutated AML Cells.

Simple SummaryOne-third of adult acute myeloid leukemia (AML) harbors NPM1 mutations. A deep knowledge of the distribution of selected antigens on the surface of NPM1-mutated AML cells may help optimizing new therapies for this frequent AML subtype. CD123 is known to be expressed on leukemic cells but also on healthy hematopoietic and endothelial cells, although at lower levels. Differences in antigen densities between AML and healthy cells may enlighten therapeutic windows, where targeting CD123 could be effective without triggering “on-target off-tumor” toxicities. Here, we perform a thorough analysis of CD123 expression demonstrating high expression of this antigen on both NPM1-mutated bulk leukemic cells and CD34+CD38− cells.NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) comprises about 30% of newly diagnosed AML in adults. Despite notable advances in the treatment of this frequent AML subtype, about 50% of NPM1mut AML patients treated with conventional treatment die due to disease progression. CD123 has been identified as potential target for immunotherapy in AML, and several anti-CD123 therapeutic approaches have been developed for AML resistant to conventional therapies. As this antigen has been previously reported to be expressed by NPM1mut cells, we performed a deep flow cytometry analysis of CD123 expression in a large cohort of NPM1mut and wild-type samples, examining the whole blastic population, as well as CD34+CD38− leukemic cells. We demonstrate that CD123 is highly expressed on NPM1mut cells, with particularly high expression levels showed by CD34+CD38− leukemic cells. Additionally, CD123 expression was further enhanced by FLT3 mutations, which frequently co-occur with NPM1 mutations. Our results identify NPM1-mutated and particularly NPM1/FLT3 double-mutated AML as disease subsets that may benefit from anti-CD123 targeted therapies.

Genetic biomarkers identify a subgroup of high-risk patients within low-risk NPM1-mutated acute myeloid leukemia

Although acute myeloid leukemia (AML) with NPM1 mut /FLT3-ITDneg is a low-risk entity, its relapse rate remains high. Out of 333 AML patients, 27 were NPM1 mut, and were analyzed in greater detail in order to find associations between clinical and molecular features and cumulative incidence of relapse. Next-generation sequencing (NGS) was performed on diagnosis and remission samples using two capture-based panels. The presence of the FLT3 D835 variant at diagnosis and a qPCR value of NPM1 mut ≥0.1% after induction chemotherapy were associated with an increased probability of relapse, especially if both conditions are present together. By contrast, patients in which the main clone found at diagnosis harbored NPM1 variant had a lower risk of relapse. Nineteen of the 85 variants found at diagnosis were detected by NGS in remission. AML Subgroup with NPM1 mut/FLT3-ITDneg is a heterogeneous entity, which can be further risk-stratified based on molecular biomarkers.

Impact of gemtuzumab ozogamicin on MRD and relapse risk in patients with NPM1-mutated AML: results from the AMLSG 09-09 trial

AbstractMonitoring of measurable residual disease (MRD) provides prognostic information in patients with Nucleophosmin1-mutated (NPM1mut) acute myeloid leukemia (AML) and represents a powerful tool to evaluate treatment effects within clinical trials. We determined NPM1mut transcript levels (TLs) by quantitative reverse-transcription polymerase chain reaction and evaluated the prognostic impact of NPM1mut MRD and the effect of gemtuzumab ozogamicin (GO) on NPM1mut TLs and the cumulative incidence of relapse (CIR) in patients with NPM1mut AML enrolled in the randomized phase 3 AMLSG 09-09 trial. A total of 3733 bone marrow (BM) samples and 3793 peripheral blood (PB) samples from 469 patients were analyzed. NPM1mut TL log10 reduction ≥ 3 and achievement of MRD negativity in BM and PB were significantly associated with a lower CIR rate, after 2 treatment cycles and at end of treatment (EOT). In multivariate analyses, MRD positivity was consistently revealed to be a poor prognostic factor in BM and PB. With regard to treatment effect, the median NPM1mut TLs were significantly lower in the GO-Arm across all treatment cycles, resulting in a significantly greater proportion of patients achieving MRD negativity at EOT (56% vs 41%; P = .01). The better reduction in NPM1mut TLs after 2 treatment cycles in MRD positive patients by the addition of GO led to a significantly lower CIR rate (4-year CIR, 29.3% vs 45.7%, P = .009). In conclusion, the addition of GO to intensive chemotherapy in NPM1mut AML resulted in a significantly better reduction in NPM1mut TLs across all treatment cycles, leading to a significantly lower relapse rate.

NPM1-Mutated Myeloid Neoplasms with

Nucleophosmin (NPM1) gene mutations rarely occur in non-acute myeloid neoplasms (MNs) with <20% blasts. Among nearly 10,000 patients investigated so far, molecular analyses documented NPM1 mutations in around 2% of myelodysplastic syndrome (MDS) cases, mainly belonging to MDS with excess of blasts, and 3% of myelodysplastic/myeloproliferative neoplasm (MDS/MPN) cases, prevalently classified as chronic myelomonocytic leukemia. These uncommon malignancies are associated with an aggressive clinical course, relatively rapid progression to overt acute myeloid leukemia (AML) and poor survival outcomes, raising controversies on their classification as distinct clinico-pathologic entities. Furthermore, fit patients with NPM1-mutated MNs with <20% blasts could benefit most from upfront intensive chemotherapy for AML rather than from moderate intensity MDS-directed therapies, although no firm conclusion can currently be drawn on best therapeutic approaches, due to the limited available data, obtained from small and mainly retrospective series. Caution is also suggested in definitely diagnosing NPM1-mutated MNs with blast count <20%, since NPM1-mutated AML cases frequently present dysplastic features and multilineage bone marrow cells showing abnormal cytoplasmic NPM1 protein delocalization by immunohistochemical staining, therefore belonging to NPM1-mutated clone regardless of blast morphology. Further prospective studies are warranted to definitely assess whether NPM1 mutations may become sufficient to diagnose AML, irrespective of blast percentage.

NPM1 mutant maintains ULK1 protein stability via TRAF6-dependent ubiquitination to promote autophagic cell survival in leukemia.

Nucleophosmin (NPM1) mutations are the most frequent genetic alteration in acute myeloid leukemia (AML) and aberrant cytoplasm-dislocated NPM1 mutant is a distinct biological characterization of this disease. Our group previously reported that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival. However, the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated. Here, we showed that Unc-51-like kinase 1 (ULK1) as a core autophagy protein was highly expressed in NPM1-mA positive OCI-AML3 cells and primary NPM1-mutated AML blasts. Meanwhile, we found that NPM1-mA could interact with ULK1 protein and positively regulated ULK1 protein levels. Mechanically, NPM1-mA promoted TRAF6-dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity via miR-146a. In addition, ULK1 high expression-mediated autophagy activation and facilitated to leukemic cell proliferation. Finally, we demonstrated that restoring ULK1 expression, ULK1 inhibitor SBI-0206965 treatment and using shULK1 partially rescued the effect of NPM1-mA on autophagy and cell survival. In conclusion, our findings suggest that NPM1 mutant interacts with ULK1, and thus, maintains its protein stability, which is required for NPM1 mutant-mediated autophagic cell survival. These data extend our understanding of the functions of NPM1 mutant in the regulation of autophagy activation in NPM1-mutated AML.

Oncogenic role of lncRNA CRNDE in acute promyelocytic leukemia and NPM1-mutant acute myeloid leukemia

The PML/RARα fusion protein acts in concert with cooperative genetic events in the development of acute promyelocytic leukemia (APL). However, oncogenic long non-coding RNAs (lncRNAs) cooperating with PML/RARα remain under-explored. Here, we first identified a set of pathogenesis-related lncRNAs, aberrantly expressed in APL using RNA-seq data from a large cohort of acute myeloid leukemia (AML) patients and normal counterparts. Among the pathogenesis-related lncRNAs, one of the evolutionarily conservative lncRNAs CRNDE (Colorectal Neoplasia Differentially Expressed) drew our attention. We found that CRNDE was highly expressed in the disease state but not in the preleukemic stage of APL, suggesting that CRNDE might be a secondary event coordinating with PML/RARα to promote APL development. Functional analysis showed that CRNDE knockdown induced differentiation and inhibited proliferation of APL cells, and prolonged survival of APL mice. Further mechanistic studies showed that CRNDE elicited its oncogenic effects through binding the miR-181 family and thereby regulating NOTCH2. Finally, we found that high CRNDE expression was also significantly correlated with NPM1 mutations and contributed to the differentiation block in NPM1-mutant AML. Collectively, our findings shed light on the importance of oncogenic lncRNAs in the development of AML and provide a promising target for AML therapy.

CD200 expression marks leukemia stem cells in human AML

AbstractThe leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34– and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34–, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.

Assay Specificity and Sensitivity Evaluation of Cepheid GeneXpert® NPM1 Prototype for Mutation Detection in Acute Myeloid Leukemia

Background: Mutations in thenucleophosmin1 (NPM1) are considered as founder mutations in acute myeloid leukemia (AML). NPM1 mutations in exon 12 occur in about 60% adult AML patients with normal karyotype. Among all NPM1 subtypes, type A (69.15%), type B (7.41%) and type D (7.41%) are found to be most frequent. The evaluation of minimal residual disease (MRD) has been considered critical to more precisely define AML response to clinical treatment, thereby refining disease risk stratification. Currently, AML with NPM1 gene mutations is recognized as a distinct entity, due to its unique clinical features. A large body of evidence demonstrated a significant independent prognosis factor of molecular MRD monitoring in NPM1-mutated AML. Objectives: Here we present GeneXpert NPM1 prototype, a fast and promising test for use in AML mutation detection (this prototype is for research use only, not for use in diagnostic procedures, and not reviewed by any regulatory body). NPM1 prototype is an easy-to-use-sample in/results out, and highly sensitive assay. The turnaround time is as fast as less than 3 hours. NPM1 prototype is designed for the quantitative detection of NPM1 mutant mRNA transcripts (types A, B and D in exon 12) and the ABL endogenous control mRNA transcript in peripheral blood of AML patients. This test is also designed for use as an aid in the monitoring of mutant NPM1 mRNA transcript levels in patients diagnosed with NPM1-mutated AML. This test does not differentiate between A, B or D type mutant NPM1 transcripts and does not detect or monitor other rare types of mutant NPM1. Results: Through assay optimization, we were able to reach limit of detection (LoD) within 0.05% for three NPM1 subtypes among all screened EDTA donor blood samples. Ct-copy number regression curve aligns well among three subtypes, within detection range. Our assay specificity has been above 95% among 30 screened EDTA donor blood samples. Conclusion: In summary, GeneXpert NPM1 prototype shows promise as a fast test for use in AML mutation detection.The presented NPM1 prototype has a great advantage covering most prevalent NPM1 mutation types up to 94% of all NPM1 subtypes. Our results demonstrated that our prototype shows satisfactory assay specificity and sensitivity. Clinical samples will be included for our clinical validation in the future. Disclosures No relevant conflicts of interest to declare.

Meta-Analysis of Genome-Wide Association Studies of Acute Myeloid Leukemia (AML) Patients Identifies Variants Associated with Risk of 11q23/KMT2A-Translocated and Core-Binding Factor (CBF) AML and Suggests a Role for Transcription Elongation in Leukemogenesis

Meta-Analysis of Genome-Wide Association Studies of Acute Myeloid Leukemia (AML) Patients Identifies Variants Associated with Risk of 11q23/KMT2A-Translocated and Core-Binding Factor (CBF) AML and Suggests a Role for Transcription Elongation in Leukemogenesis

Routine Use of Gemtuzumab Ozogamicin in 7+3-Based Inductions for All “Non-Adverse” Risk AML

Introduction: The addition of gemtuzumab ozogamicin (GO) to 7+3 chemotherapy for newly diagnosed acute myeloid leukemia (AML) has been shown to significantly improve event-free survival (EFS) for cytogenetically favorable-risk AML, with marginal benefit for intermediate-risk AML, and no benefit for cytogenetically adverse-risk AML. Of note, with the exception of mutated FLT3-ITD, little is known about the impact of GO in ELN 2017-defined genotypically adverse-risk AML, and a recent randomized trial found no EFS benefit for 7+3+GO in patients (pts) with genotypically favorable-risk, NPM1-mutated AML. Since 2017, our institution incorporated GO into 7+3-based inductions for all "non-adverse" risk AML pts, as defined by wild-type FLT3 and no abnormalities on rapid FISH analysis for del(5q)/monosomy 5, del(7q)/monosomy 7, and del(20q). We report our experience treating all pts with "non-adverse" risk AML-as defined by this algorithm-with 7+3+GO. Methods: An institutional database was queried in order to identify all pts ≥18 years old who received 7+3-based chemotherapy for newly diagnosed AML between 2017 and 2020; pts who received the FDA-approved fractionated dose of GO were included in the analysis. Data collection included demographic variables, karyotype/FISH, targeted PCR analyses, and multigene NGS panels for AML-related mutations including, but not limited to, mutations in FLT3, NPM1, CEBPA, TP53, RUNX1, and ASXL1. Outcome data included response to induction, relapse, and death, as well as hematopoietic cell transplant (HCT) rates, conditioning regimens, and post-transplant complications. Results: Between January 2017 and July 2020, 96 pts received 7+3-based induction at our institution. Of these, 29 (30%) received 7+3 in combination with GO. Median age at diagnosis was 46 years (range 23-66), with 17 (59%) males. Sixteen (55%) pts had ELN favorable-risk AML (5 [31%] by cytogenetics and 11 [69%] by genotype), 6 (21%) pts had ELN intermediate-risk AML, and 7 (24%) pts had ELN adverse-risk AML (4 [57%] by cytogenetics and 3 [43%] by genotype). Median time from diagnosis to start of induction was 4 days (range 0-43). For cytogenetically adverse-risk pts, median time from diagnostic bone marrow biopsy to receipt of adverse karyotype results was 8 days (7-14). Median time from start of induction to receipt of multigene NGS results for all pts was 15 days (3-32). Overall, 22 (76%) pts achieved remission. All genotypically adverse-risk pts (1 with mutated TP53 and 2 with mutated RUNX1) were refractory to induction, while 3 of 4 (75%) cytogenetically adverse-risk pts (1 with t(6;9), 1 with monosomy 7, and 2 with 11q23 abnormalities) achieved remission. Eight of the 29 (28%) pts proceeded to HCT, including 4 adverse-risk pts. Of the adverse-risk pts, all received myeloablative conditioning prior to HCT and 3 (75%) developed veno-occlusive disease (VOD), with 2 (50%) requiring defibrotide therapy. In favorable/intermediate-risk pts, 4 (18%) proceeded to HCT (2 intermediate-risk pts in first remission and 2 favorable-risk pts in second remission). Of these, 2 (50%) received myeloablative conditioning and 1 (25%) developed VOD. At last follow-up, 23 of 29 pts (79%) remained alive, with a median overall survival not reached (range 1-29 months) and a median EFS of 20 months (9-31). The percentage of ELN favorable-, intermediate-, and adverse-risk pts who remained event-free at last follow-up was 75%, 33%, and 43%, respectively. Discussion: This single-center, retrospective cohort describes the outcomes of pts with "non-adverse" risk AML who received induction chemotherapy with 7+3+GO according to a pre-defined algorithm. Using this algorithm, 30% of all pts receiving 7+3-based inductions received GO. Of these, nearly 25% were ultimately found to have adverse-risk AML as defined by ELN 2017 criteria, largely driven by long turn-around times for karyotyping and NGS multigene panel results. No patient with genotypically adverse-risk AML by ELN criteria responded to induction chemotherapy, and 75% of cytogenetically adverse-risk pts who proceeded to HCT developed VOD. Routine use of 7+3+GO induction outside of the context of cytogenetically favorable-risk AML remains controversial, and further study is needed to define the role of GO, particularly for pts with ELN genotypically adverse-risk AML. Table Disclosures Gotlib: Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding. Liedtke:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; GSK: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees; Caelum: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Muffly:Adaptive: Research Funding; Amgen: Consultancy; Servier: Research Funding. Mannis:AbbVie, Agios, Bristol-Myers Squibb, Genentech: Consultancy; Glycomimetics, Forty Seven, Inc, Jazz Pharmaceuticals: Research Funding.

Clonal Hematopoiesis and Its Implications for Flow Cytometric Assessment of Measurable Residual Disease in Patients with NPM1-mutated Acute Myeloid Leukemia

Clonal Hematopoiesis and Its Implications for Flow Cytometric Assessment of Measurable Residual Disease in Patients with NPM1-mutated Acute Myeloid Leukemia

DNA-Based Methods for Measurable Residual Disease Detection in NPM1-Mutated Acute Myeloid Leukemia; Establishment of Cut-Offs for qPCR, Digital Droplet PCR and Targeted Deep Sequencing

For detection of measurable residual disease (MRD) in acute myeloid leukemia with NPM1 mutations, RT-qPCR with quantification of leukemic transcripts is currently considered the method of choice; however, MRD can also be determined with DNA-based methods, offering certain advantages. For example, digital droplet PCR (ddPCR) and targeted deep sequencing (deep seq) do neither require standard curves nor reference genes and are thus less labor intense than RT-qPCR. Also, deep seq allows for quantification independently of type of NPM1 mutation. In addition, DNA-based techniques enable MRD assessment of other mutations, beyond the reach of RT-qPCR, which is limited to analyses of highly expressed genes or fusion transcripts (e.g. core binding factor leukemias). With the rapid development of highly sensitive DNA-based techniques for MRD detection, there is a need to establish clinically relevant cut-offs for accurate interpretation of MRD results and risk stratification. Here, we compare and provide MRD cut-offs for three different DNA-based MRD methods for NPM1 mutations: quantitative PCR (qPCR), ddPCR and deep seq. To compare the DNA-based methods with RT-qPCR, we analyzed 110 follow-up peripheral blood (PB) or bone marrow (BM) samples from 32 AML patients harboring NPM1 mutation type A. First, we compared the mere detectability of leukemic signals (without reference to specific MRD cut-off points). We found a high correlation between results from RT-qPCR and the three DNA-based methods (Rs=0.936 for RT-qPCR vs qPCR, Rs=0.774 for RT-qPCR vs ddPCR and Rs=0.743 for RT-qPCR vs deep seq, p&amp;lt;0.001). As expected, RT-qPCR was the most sensitive method. Among the DNA-based methods, qPCR was the most sensitive, detecting leukemic DNA in 95% (55/58) of the RT-qPCR positive samples, compared to 72% (42/58) and 62% (36/58) for ddPCR and deep seq, respectively. Interestingly, the transcript level for a given amount of measurable leukemic DNA (RNA copy number per leukemic DNA molecule) fluctuated considerably between different follow-up samples for certain patients. In some cases, the RNA/DNA ratio exceeded a hundredfold difference between different follow-up time points in both PB and BM. Hence, transcript analysis may be more complex than just a simple measurement of leukemic cell burden, which in turn may influence accurate risk stratification and treatment decisions, if relying on RT-qPCR measurements alone. To select adequate DNA MRD cut-offs, we performed ROC curve analyses for each method at various DNA cut-offs, comparing them with the gold standard RT-qPCR cut-off. In BM, this cut-off can be defined as a less than 3 log reduction of mutated NPM1 transcripts vs diagnosis, separating MRDhigh from MRDlow/undetectable (sometimes inaccurately termed "MRD-positivity" and "MRD-negativity", respectively). In PB, the mere detectability of mutated NPM1 transcripts is considered the relevant cut-off. DNA cut-offs were chosen based on the area under the curve (AUC) for the ROC analyses (Table 1), and influenced by available literature including recommendations of prognostically relevant MRD levels. For qPCR, a cut-off at 0.1% leukemic DNA was judged relevant in BM. For ddPCR and deep seq, 0.05% was chosen to adjust for measuring allelic ratio (variant allele frequency (VAF)) rather than mutant DNA alone. In PB, the selected cut-off was detectable leukemic signal irrespective of DNA method. We next determined the accuracy of the selected cut-offs, for identification of samples with clinically relevant MRD, by comparing them with the gold standard RT-qPCR. In general, the selected DNA cut-off values generated high specificity as well as high positive and negative predictive values (Table 1). The vast majority of all MRD analyses (93% (368/395)) showed concordant results irrespective of MRD method. In BM samples, MRD assessment by the DNA based methods agreed with MRD status as determined by RT-qPCR (MRDhigh high vs MRDlow/undetectable) in 93% (62/67) of the analyses for qPCR, 96% (64/67) for ddPCR, and 97% (65/67) for deep seq. In PB, the agreement was 95% (41/43), 88% (38/43) and 86% (37/43), respectively. In summary, we found strong agreement between different MRD methods and based on this could provide clinically relevant cut-offs for risk stratification. Thus, in BM follow-up samples from AML patients with NPM1 mutation, we propose 0.1% leukemic DNA as cut-off for qPCR and 0.05% VAF for ddPCR and deep seq. Disclosures No relevant conflicts of interest to declare.

The Natural History of NPM1MUT Measurable Residual Disease (MRD) Positivity after Completion of Chemotherapy in Acute Myeloid Leukemia (AML)

The Natural History of NPM1MUT Measurable Residual Disease (MRD) Positivity after Completion of Chemotherapy in Acute Myeloid Leukemia (AML)