Advantages of a genomic DNA-based next-generation sequencing assay for detection of mutant NPM1 measurable residual disease in AML

Mutations in the nucleophosmin-1 (NPM1) gene are among the most common molecular aberrations in acute myeloid leukemia (AML). Various studies have established mutant NPM1 (mNPM1) as a faithful molecular measurable residual disease (MRD) marker with prognostic significance. Assessment of prognostic mNPM1 is included in the European LeukemiaNet recommendations on MRD detection in AML. Because of recent advancements of promising drugs targeting mNPM1 AML, monitoring of mNPM1 MRD has gained interest, and is generally done by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). However, these RT-qPCR assays use complementary DNA (cDNA) as input, are based on gene expression levels of mNPM1, and are generally limited to specific mNPM1 gene variants. The main advantages of next-generation sequencing (NGS) using genomic DNA as input are stability, independence of gene expression levels, and the ability to detect any NPM1 variant in a single assay. Here, we comprehensively investigated the applicability of NGS on DNA to detect mNPM1 MRD in a cohort of 119 (cDNA) and 310 (DNA) patients with mNPM1 AML in complete remission after 2 cycles of induction chemotherapy. We demonstrate high correlations in levels and prognostic value between RT-qPCR/cDNA and NGS/DNA approaches, postulating NGS/DNA as an attractive alternative to RT-qPCR. We report that the 2% mNPM1/ABL1 threshold by RT-qPCR/cDNA corresponds to an NGS/DNA variant allele frequency of 0.01%. The NGS/DNA threshold of &gt;0.01% after 2 cycles of induction chemotherapy identifies significantly more patients with AML with an increased relapse risk than current RT-qPCR/cDNA assays. The prognostic significance of mNPM1 MRD appears greatest in patients with AML with FLT3-internal tandem duplications.</p

Advantages of a genomic DNA-based NGS assay for detection of mutant NPM1 measurable residual disease in AML

Mutations in the Nucleophosmin-1 (NPM1) gene are among the most common molecular aberrations in acute myeloid leukemia (AML). Various studies have established mutant NPM1 (mNPM1) as a faithful molecular measurable residual disease (MRD) marker with prognostic significance. Assessment of prognostic mNPM1 is included in the European LeukemiaNet (ELN) recommendations on MRD detection in AML. Due to recent advancements of promising drugs targeting mNPM1 AML, monitoring of mNPM1 MRD has gained interest, and is generally done by reverse transcriptase quantitative PCR (RT-qPCR). However, these RT-qPCR assays use cDNA as input, are based on gene expression levels of mNPM1, and are generally limited to specific mNPM1 gene variants. The main advantages of next-generation sequencing (NGS) using genomic DNA as input are stability, independence of gene expression levels, and the ability to detect any NPM1 variant in a single assay. Here we comprehensively investigated the applicability of NGS on DNA to detect mNPM1 MRD in a cohort of 119 (cDNA) and 310 (DNA) mNPM1 AML patients in complete remission after two cycles of induction chemotherapy. We demonstrate high correlations in levels and prognostic value between RT-qPCR/cDNA and NGS/DNA approaches, postulating NGS/DNA as an attractive alternative to RT-qPCR. We report that the 2% mNPM1/ABL1 threshold by RT-qPCR/cDNA corresponds to a NGS/DNA variant allele frequency (VAF) of 0.01%. The NGS/DNA threshold of &gt;0.01% after two cycles of induction chemotherapy identifies significantly more AML patients with an increased relapse risk than current RT-qPCR/cDNA assays. The prognostic significance of mNPM1 MRD appears greatest in FLT3-ITD AML patients.</p

Advantages of a genomic DNA-based next-generation sequencing assay for detection of mutant NPM1 measurable residual disease in AML

Mutations in the nucleophosmin-1 (NPM1) gene are among the most common molecular aberrations in acute myeloid leukemia (AML). Various studies have established mutant NPM1 (mNPM1) as a faithful molecular measurable residual disease (MRD) marker with prognostic significance. Assessment of prognostic mNPM1 is included in the European LeukemiaNet recommendations on MRD detection in AML. Because of recent advancements of promising drugs targeting mNPM1 AML, monitoring of mNPM1 MRD has gained interest, and is generally done by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). However, these RT-qPCR assays use complementary DNA (cDNA) as input, are based on gene expression levels of mNPM1, and are generally limited to specific mNPM1 gene variants. The main advantages of next-generation sequencing (NGS) using genomic DNA as input are stability, independence of gene expression levels, and the ability to detect any NPM1 variant in a single assay. Here, we comprehensively investigated the applicability of NGS on DNA to detect mNPM1 MRD in a cohort of 119 (cDNA) and 310 (DNA) patients with mNPM1 AML in complete remission after 2 cycles of induction chemotherapy. We demonstrate high correlations in levels and prognostic value between RT-qPCR/cDNA and NGS/DNA approaches, postulating NGS/DNA as an attractive alternative to RT-qPCR. We report that the 2% mNPM1/ABL1 threshold by RT-qPCR/cDNA corresponds to an NGS/DNA variant allele frequency of 0.01%. The NGS/DNA threshold of &gt;0.01% after 2 cycles of induction chemotherapy identifies significantly more patients with AML with an increased relapse risk than current RT-qPCR/cDNA assays. The prognostic significance of mNPM1 MRD appears greatest in patients with AML with FLT3-internal tandem duplications.</p

Addition of Anti-thymocyte Globulin in Allogeneic Stem Cell Transplantation With Peripheral Stem Cells From Matched Unrelated Donors Improves Graft-Versus-Host Disease and Relapse Free Survival

Anti-thymocyte globulin (ATG) is commonly used to prevent graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). To evaluate the impact of ATG as part of the GvHD prophylaxis in our institution, we report the outcome of 415 patients with matched unrelated donors (MUD) transplanted for hematological malignancies with or without ATG from 2005 to 2019 at Oslo University Hospital, Norway. The following groups were compared: (1) 154 patients transplanted with peripheral blood stem cells (PBSC) without ATG 2005-2014. (2) 137 patients transplanted with bone marrow stem cells (BMSC) 2005-2019. (3) 124 patients transplanted with PBSC and ATG (PBSC + ATG) 2014-2019. Three years survival was similar in the groups, 61% following allografting with PBSC, 54% with BMSC, and 59% with PBSC + ATG. Acute GvHD grade III-IV was 14%, 14%, and 7%; chronic GvHD was 81%, 32, and 26%; and extensive cGvHD 44%, 15%, and 6% in the corresponding groups. Both acute and chronic GvHD were significantly reduced in the PBSC + ATG-versus the PBSC group (p < 0.05 and p < 0.001 respectively).Transplant-related mortality (TRM) was 33%, 25%, and 17% (p = 0.18). Graft versus host disease and relapse free survival (GRFS) at 3 years was 43 %, 43%, and 64% in the groups. Adding ATG to the GvHD prophylaxis regimen of MUD allo-HSCT with PBSC resulted in a substantial reduction of both acute and chronic GvHD without compromising the disease control, reflected in a superior 3 years GRFS

Bayesian interim analysis for prospective randomized studies:reanalysis of the acute myeloid leukemia HOVON 132 clinical trial

Randomized controlled trials (RCTs) are the gold standard to establish the benefit-risk ratio of novel drugs. However, the evaluation of mature results often takes many years. We hypothesized that the addition of Bayesian inference methods at interim analysis time points might accelerate and enforce the knowledge that such trials may generate. In order to test that hypothesis, we retrospectively applied a Bayesian approach to the HOVON 132 trial, in which 800 newly diagnosed AML patients aged 18 to 65 years were randomly assigned to a "7 + 3" induction with or without lenalidomide. Five years after the first patient was recruited, the trial was negative for its primary endpoint with no difference in event-free survival (EFS) between experimental and control groups (hazard ratio [HR] 0.99, p = 0.96) in the final conventional analysis. We retrospectively simulated interim analyses after the inclusion of 150, 300, 450, and 600 patients using a Bayesian methodology to detect early lack of efficacy signals. The HR for EFS comparing the lenalidomide arm with the control treatment arm was 1.21 (95% CI 0.81-1.69), 1.05 (95% CI 0.86-1.30), 1.00 (95% CI 0.84-1.19), and 1.02 (95% CI 0.87-1.19) at interim analysis 1, 2, 3 and 4, respectively. Complete remission rates were lower in the lenalidomide arm, and early deaths more frequent. A Bayesian approach identified that the probability of a clinically relevant benefit for EFS (HR &lt; 0.76, as assumed in the statistical analysis plan) was very low at the first interim analysis (1.2%, 0.6%, 0.4%, and 0.1%, respectively). Similar observations were made for low probabilities of any benefit regarding CR. Therefore, Bayesian analysis significantly adds to conventional methods applied for interim analysis and may thereby accelerate the performance and completion of phase III trials.</p

Bayesian interim analysis for prospective randomized studies:reanalysis of the acute myeloid leukemia HOVON 132 clinical trial

Randomized controlled trials (RCTs) are the gold standard to establish the benefit-risk ratio of novel drugs. However, the evaluation of mature results often takes many years. We hypothesized that the addition of Bayesian inference methods at interim analysis time points might accelerate and enforce the knowledge that such trials may generate. In order to test that hypothesis, we retrospectively applied a Bayesian approach to the HOVON 132 trial, in which 800 newly diagnosed AML patients aged 18 to 65 years were randomly assigned to a “7 + 3” induction with or without lenalidomide. Five years after the first patient was recruited, the trial was negative for its primary endpoint with no difference in event-free survival (EFS) between experimental and control groups (hazard ratio [HR] 0.99, p = 0.96) in the final conventional analysis. We retrospectively simulated interim analyses after the inclusion of 150, 300, 450, and 600 patients using a Bayesian methodology to detect early lack of efficacy signals. The HR for EFS comparing the lenalidomide arm with the control treatment arm was 1.21 (95% CI 0.81–1.69), 1.05 (95% CI 0.86–1.30), 1.00 (95% CI 0.84–1.19), and 1.02 (95% CI 0.87–1.19) at interim analysis 1, 2, 3 and 4, respectively. Complete remission rates were lower in the lenalidomide arm, and early deaths more frequent. A Bayesian approach identified that the probability of a clinically relevant benefit for EFS (HR &lt; 0.76, as assumed in the statistical analysis plan) was very low at the first interim analysis (1.2%, 0.6%, 0.4%, and 0.1%, respectively). Similar observations were made for low probabilities of any benefit regarding CR. Therefore, Bayesian analysis significantly adds to conventional methods applied for interim analysis and may thereby accelerate the performance and completion of phase III trials.</p

Early response evaluation by single cell signaling profiling in acute myeloid leukemia

Aberrant pro-survival signaling is a hallmark of cancer cells, but the response to chemotherapy is poorly understood. In this study, we investigate the initial signaling response to standard induction chemotherapy in a cohort of 32 acute myeloid leukemia (AML) patients, using 36-dimensional mass cytometry. Through supervised and unsupervised machine learning approaches, we find that reduction of extracellular-signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation in the myeloid cell compartment 24 h post-chemotherapy is a significant predictor of patient 5-year overall survival in this cohort. Validation by RNA sequencing shows induction of MAPK target gene expression in patients with high phosphoERK1/2 24 h post-chemotherapy, while proteomics confirm an increase of the p38 prime target MAPK activated protein kinase 2 (MAPKAPK2). In this study, we demonstrate that mass cytometry can be a valuable tool for early response evaluation in AML and elucidate the potential of functional signaling analyses in precision oncology diagnostics.Peer reviewe

Clinical practice recommendation on hematopoietic stem cell transplantation for acute myeloid leukemia patients with FLT3 internal tandem duplication: a position statement from the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in 25-30% of patients with acute myeloid leukemia . Because of the poor prognosis associated with FMS-like tyrosine kinase 3 internal tandem duplication mutated Acute myeloid leukemia, allogeneic-hematopoietic stem-cell transplantation was commonly performed in first complete remission. Remarkable progress has been made in frontline treatments with the incorporation of FLT3 inhibitors and the development of highly sensitive minimal/measurable residual disease assays. Similarly, recent progress in allogeneic-hematopoietic stem-cell transplantation includes improvement of transplant techniques, the use of haplo-identical donors in patients lacking an HLA matched donor, and the introduction of FLT3 inhibitors as posttransplant maintenance therapy. Nevertheless, current transplant strategies vary between centers and differ in terms of transplant indications based on the internal tandem duplication allelic ratio and concomitant nucleophosmin-1 mutation, as well as in terms of post-transplant maintenance/consolidation. This review generated by international leukemia or transplant experts, mostly from the European Society for Blood and Marrow Transplantation, attempts to develop a position statement on best approaches for allogeneic-hematopoietic stem-cell transplantation for acute myeloid leukemia with FMS-like tyrosine kinase internal tandem duplication including indications and modalities of allogeneic-hematopoietic stem-cell transplantation and on potential optimization of post-transplant maintenance with FMS-like tyrosine kinase inhibitors

A Nationwide Study of GATA2 Deficiency in Norway-the Majority of Patients Have Undergone Allo-HSCT

PurposeGATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT.MethodsAll medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records.ResultsBetween 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively.ConclusionOur main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described